**Figure 5.** CHRNA5 regulates YAP activity in HCC. (**A**) KEGG analysis of genes negatively correlated with CHRNA5 in HCC; (**B**) GO analysis of genes negatively correlated with CHRNA5 in HCC; (**C**) KEGG analysis of genes positively correlated with CHRNA5 in HCC; (**D**) GO analysis of genes positively correlated with CHRNA5 in HCC; (**E**) Western blot analysis of YAP in PLC-NC and PLC-sh CHRNA5 cell lines; (**F**) Western blot analysis of YAP in Huh7-CMV and Huh7- CHRNA5 OE cell lines; (**G**) Western blot analysis of YAP expression in cytoplasm and nucleus from PLC-NC and PLC-sh CHRNA5 cell lines; (**H**) Western blot analysis of YAP expression in cytoplasm **Figure 5.** CHRNA5 regulates YAP activity in HCC. (**A**) KEGG analysis of genes negatively correlated with CHRNA5 in HCC; (**B**) GO analysis of genes negatively correlated with CHRNA5 in HCC; (**C**) KEGG analysis of genes positively correlated with CHRNA5 in HCC; (**D**) GO analysis of genes positively correlated with CHRNA5 in HCC; (**E**) Western blot analysis of YAP in PLC-NC and PLC-sh CHRNA5 cell lines; (**F**) Western blot analysis of YAP in Huh7-CMV and Huh7-CHRNA5 OE cell lines; (**G**) Western blot analysis of YAP expression in cytoplasm and nucleus from PLC-NC and PLC-sh CHRNA5 cell lines; (**H**) Western blot analysis of YAP expression in cytoplasm and nucleus from Huh7-CMV and Huh7-CHRNA5 OE cell lines.

#### *3.6. YAP Plays an Essential Role in the Contribution of CHRNA5 to Malignant Phenotype of HCC*

*3.6. YAP Plays an Essential Role in the Contribution of CHRNA5 to Malignant Phenotype of HCC*  To uncover whether YAP plays an essential role in the contribution of CHRNA5 to the HCC malignant phenotype, we overexpressed YAP in CHRNA5-silencing PLC cell lines and silenced YAP expression in CHRNA5-overexpressing Huh7 cell lines. The results revealed that YAP overexpression partially reversed the inhibitory effect of silencing CHRNA5 on the migration ability and expression of EMT-associated markers (Figure 6A,B).

and nucleus from Huh7-CMV and Huh7-CHRNA5 OE cell lines.

Furthermore, the inhibitory effects of silencing CHRNA5 on the tumor spheroid formation ability of PLC cell lines and the expression of stemness-associated genes were also attenuated after overexpressing YAP (Figure 6C,D). Accordingly, the opposite effect was observed after silencing YAP in CHRNA5-overexpressing Huh7 cell lines (Figure 6E–H). These results suggest that CHRNA5 can regulate the malignant phenotype of HCC cell lines by modulating YAP activity. (Figure 6A,B). Furthermore, the inhibitory effects of silencing CHRNA5 on the tumor spheroid formation ability of PLC cell lines and the expression of stemness-associated genes were also attenuated after overexpressing YAP (Figure 6C,D). Accordingly, the opposite effect was observed after silencing YAP in CHRNA5-overexpressing Huh7 cell lines (Figure 6E–H). These results suggest that CHRNA5 can regulate the malignant phenotype of HCC cell lines by modulating YAP activity.

To uncover whether YAP plays an essential role in the contribution of CHRNA5 to the HCC malignant phenotype, we overexpressed YAP in CHRNA5-silencing PLC cell lines and silenced YAP expression in CHRNA5-overexpressing Huh7 cell lines. The results revealed that YAP overexpression partially reversed the inhibitory effect of silencing CHRNA5 on the migration ability and expression of EMT-associated markers

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 11 of 16

**Figure 6.** YAP plays an essential role in the contribution of CHRNA5 to malignant phenotype of HCC. (**A**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the migration ability of HCC cells; (**B**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the expression of EMT-associated markers; (**C**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the spheroid formation ability of HCC cells; (**D**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the expression of stemness-associated genes; (**E**) the inhibitory effect of silencing CHRNA5 on the migration ability of HCC cells was attenuated after overexpressing YAP; (**F**) the inhibitory effect of silencing CHRNA5 on the expression of EMT-associated markers in HCC cells was attenuated after **Figure 6.** YAP plays an essential role in the contribution of CHRNA5 to malignant phenotype of HCC. (**A**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the migration ability of HCC cells; (**B**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the expression of EMT-associated markers; (**C**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the spheroid formation ability of HCC cells; (**D**) YAP overexpression partially rescued the inhibitory effect of silencing CHRNA5 on the expression of stemness-associated genes; (**E**) the inhibitory effect of silencing CHRNA5 on the migration ability of HCC cells was attenuated after overexpressing YAP; (**F**) the inhibitory effect of silencing CHRNA5 on the expression of EMT-associated markers in HCC cells was attenuated after overexpressing YAP; (**G**) the inhibitory effect of silencing CHRNA5 on the spheroid formation ability of HCC cells was attenuated after overexpressing YAP; (**H**) the inhibitory effect of silencing CHRNA5 on the expression of stemness-associated genes in HCC cells was attenuated after overexpressing YAP. \* *p* < 0.05.

#### *3.7. CHRNA5 Regulates Sorafenib Sensitivity in HCC 3.7. CHRNA5 Regulates Sorafenib Sensitivity in HCC*

overexpressing YAP. \* *p* < 0.05.

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 12 of 16

Several studies have already uncovered that YAP activity is closely associated with sorafenib resistance in HCC [29,30]. In addition, stemness properties were also reported to share a close association with sorafenib sensitivity in HCC [31]. Therefore, we further evaluated the effect of CHRNA5 on HCC sensitivity to sorafenib. From the TCGA database, we observed that a higher expression level of CHRNA5 was correlated with a worse OS in HCC patients receiving sorafenib treatment (Figure 7A), suggesting that CHRNA5 might be associated with sorafenib sensitivity in HCC. An in vitro assay also revealed that silencing CHRNA5 significantly decreased the IC50 of HCC cells (Figure 7B,C), which was markedly increased when CHRNA5 was overexpressed (Figure 7D,E). The TUNEL assay also revealed that CHRNA5 silencing augmented the apoptosis of HCC cells induced by sorafenib, whereas CHRAN5 overexpression attenuated the apoptosis of HCC cells induced by sorafenib (Figure 7F,G). These results suggest that CHRNA5 contributes to sorafenib resistance in HCC and has the potential to serve as an indicator for predicting sorafenib sensitivity in HCC patients. Several studies have already uncovered that YAP activity is closely associated with sorafenib resistance in HCC [29,30]. In addition, stemness properties were also reported to share a close association with sorafenib sensitivity in HCC [31]. Therefore, we further evaluated the effect of CHRNA5 on HCC sensitivity to sorafenib. From the TCGA database, we observed that a higher expression level of CHRNA5 was correlated with a worse OS in HCC patients receiving sorafenib treatment (Figure 7A), suggesting that CHRNA5 might be associated with sorafenib sensitivity in HCC. An in vitro assay also revealed that silencing CHRNA5 significantly decreased the IC50 of HCC cells (Figure 7B,C), which was markedly increased when CHRNA5 was overexpressed (Figure 7D,E). The TUNEL assay also revealed that CHRNA5 silencing augmented the apoptosis of HCC cells induced by sorafenib, whereas CHRAN5 overexpression attenuated the apoptosis of HCC cells induced by sorafenib (Figure 7F,G). These results suggest that CHRNA5 contributes to sorafenib resistance in HCC and has the potential to serve as an indicator for predicting sorafenib sensitivity in HCC patients.

overexpressing YAP; (**G**) the inhibitory effect of silencing CHRNA5 on the spheroid formation ability of HCC cells was attenuated after overexpressing YAP; (**H**) the inhibitory effect of silencing CHRNA5 on the expression of stemness-associated genes in HCC cells was attenuated after

**Figure 7.** CHRNA5 regulates sorafenib sensitivity in HCC. (**A**) The effect of CHRNA5 on the OS of HCC patients receiving sorafenib treatment; (**B**,**C**) IC50 of sorafenib in PLC-NC and PLC-sh-CHRNA5 cell lines; (**D**,**E**) IC50 of sorafenib in Huh7-CMV and Huh7-CHRNA5 OE cell lines; (**F**) **Figure 7.** CHRNA5 regulates sorafenib sensitivity in HCC. (**A**) The effect of CHRNA5 on the OS of HCC patients receiving sorafenib treatment; (**B**,**C**) IC50 of sorafenib in PLC-NC and PLC-sh-CHRNA5 cell lines; (**D**,**E**) IC50 of sorafenib in Huh7-CMV and Huh7-CHRNA5 OE cell lines; (**F**) TUNEL detection of PLC-NC and PLC-shCHRNA5 cell lines treated with sorafenib; (**G**) TUNEL detection of Huh7-CMV and Huh7-CHRNA5 OE cell lines treated with sorafenib. \* *p* < 0.05, + Censored Data.

#### **4. Discussion**

CHRNA5, a member of the superfamily of ligand-gated ion channels, is a vital receptor for nicotine [32]. Thus, previous studies regarding CHRNA5 have largely focused on its

role in nicotine dependence and lung cancer progression [33]. In this study, for the first time, we investigated the role of CHRNA5 in HCC. The results reveal that CHRNA5 expression level is upregulated in HCC tissues and is closely associated with tumor T stage, AJCC phase, and patient prognosis. These results suggest the involvement of CHRNA5 in HCC progression. The role of CHRNA5 in tumor proliferation was previously reported. In oral squamous cell carcinoma, CHRNA5 was reported to regulate the activity of the E2F signaling pathway, a critical regulator of cell cycle and stemness properties (33986804), contributing to tumor growth and treatment resistance [24,25]. Ozlen Konu et al. also reported that CHRNA5 silencing in breast cancer significantly inhibited tumor growth, which might be attributed to the significant inhibition of cell-cycle-associated genes after CHRNA5 silencing [34]. Their study suggested that CHRNA5 might regulate cell cycle arrest during the G1 phase by modulating the activity of retinoblastoma family proteins (RB), which regulate E2F1 activity by binding with E2F1 [35]. To further validate the functional role of CHRNA5 in HCC, we induced lentivirus-mediated overexpression or silencing of CHRNA5. We found that CHRNA5 is crucial for the proliferation of HCC cells both in vitro and in vivo, consistent with the conclusion of previous studies in other types of cancers. Tumor invasion and metastasis are challenges in the clinical treatment of HCC. EMT is regarded as a critical step in tumor invasion and metastasis [36]. A previous study suggested that CHRNA5 could regulate the migration and invasion ability of melanoma cells [23]. Similarly, our study indicated that CHRNA5 could enhance the invasion and metastasis ability of HCC by regulating EMT-associated genes. All of these data suggest that CHRNA5 plays a critical role in the malignant phenotype of HCC.

Sorafenib is a multitarget molecular drug and is one of the main treatment strategies for advanced HCC. By suppressing the activity of various receptor tyrosine kinase and VEGF/Raf/MER/ERK-mediated multiple signaling pathways, sorafenib can inhibit tumor cell proliferation and angiogenesis in vivo [37]. Despite the fact that sorafenib can extend the patient's overall survival, limited benefits were observed in more than 70% of patients with advanced HCC as a result of drug resistance. Therefore, it is clinically meaningful to explore the mechanisms of sorafenib resistance in HCC patients. Our results revealed that CHRNA5 silencing could augment sorafenib sensitivity, and CHRNA5 overexpression could attenuate sorafenib sensitivity, indicating the regulatory role of CHRNA5 in sorafenib sensitivity in HCC. Accordingly, among patients receiving sorafenib treatment, those with a higher expression level of CHRNA5 exhibited a worse OS compared with those with a lower expression level of CHRNA5. These results indicate that CHRNA5 has a pivotal role in sorafenib resistance in HCC. There is a growing amount of evidence indicating that stemness properties, closely associated with cancer initiation and progression, also play a significant role in treatment resistance [38]. The maintenance of stemness properties relies on the activation of stemness-associated genes, such as Sox2, OCT4, and Nanog [38]. Moreover, several stemness-associated signaling pathways, such as Wnt/β-catenin signaling, Notch signaling, and JAK/STAT signaling, also greatly contribute to stemness properties in HCC [39]. In HCC, stemness properties are closely associated with drug resistance, tumor metastasis, and recurrence [39]. Stemness properties are closely associated with the EMT phenotype, which was previously reported to contribute to sorafenib resistance in HCC [40,41]. Several other studies also reported that stemness properties are closely associated with sorafenib resistance in HCC [42]. Ozlen Konu et al. observed a close association between CHRNA5 and Wnt/β-catenin signaling, from which we can propose that CHRNA5 might also regulate the stemness properties of breast cancer cells [34]. Consistent with their study, our study also indicated that CHRNA5 could maintain the stemness properties of HCC cells by regulating stemness-associated genes, to which the regulatory role of CHRNA5 in sorafenib sensitivity could be partially attributed. Stemness properties are also closely associated with chemotherapy resistance and radiotherapy resistance [43]. Thus, it is possible that CHRNA5 might also contribute to resistance to chemotherapy and radiotherapy in HCC, but further experiments are needed to test this hypothesis. In fact, previous studies have already identified the role of CHRNA5 in the resistance to

chemotherapy in breast cancer and the resistance to radiotherapy in oral squamous cell carcinoma [24].

YAP, playing an essential role in cancer progression, was reported to be implicated in modulating various malignant phenotypes of cancer cells, such as cell proliferation, migration, invasion, apoptosis resistance, EMT phenotype, and stemness properties [44–46]. In this study, data from the TCGA database exhibited a close association between CHRNA5 and the Hippo signaling pathway in HCC. Further in vitro assays revealed that CHRNA5 could regulate YAP activity, and YAP silencing was sufficient to reverse the CHRNA5 mediated tumor-promoting effect. Thus, these results suggest that CHRNA5 promotes HCC progression by modulating YAP activity. Recent studies also suggested the contribution of YAP to drug sensitivity in HCC through various mechanisms. Yuan Zhou et al. reported that YAP contributes to chemotherapy resistance via regulating the Rac family small guanosine triphosphatase 1 (RAC1)—reactive oxygen species (ROS)—mTOR signaling pathway [47]. Darko Castven et al. reported that YAP induced stem-like properties and accounted for the acquired resistance to sorafenib in HCC cells [48]. Ruize Gao et al. also reported that YAP could transcriptionally initiate the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, causing resistance to sorafenib-induced ferroptosis in HCC [29]. Consistent with previous studies, our study reveals that downregulation of YAP activity caused by CHRNA5 silencing leads to enhanced sensitivity to sorafenib. Thus, YAP plays a critical role in the contribution of CHRNA5 to stemness properties, EMT phenotype, and sorafenib resistance in HCC.

#### **5. Conclusions**

In conclusion, our study demonstrates that CHRNA5 plays a significant role in promoting the malignant phenotype of HCC by regulating YAP activity. Its role in sorafenib resistance suggests the potential of CHRNA5 to serve as an indicator for sorafenib sensitivity, and targeting CHRNA5 might be a strategy for HCC treatment.

**Author Contributions:** Study design, Q.D., H.J. and Y.F.; acquisition and analysis of data, Y.F., H.C., W.D., H.J. and Q.D.; drafting of the manuscript, Y.F., H.C. and W.D.; revision of the manuscript H.J. and Q.D.; Y.F. and H.C. are co-first authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (81872356), the Program of Shanghai Academic Research Leader (20XD1400900) and the Project of Medical Engineering, Fudan University (yg2021-017).

**Institutional Review Board Statement:** The animal study was performed in accordance with the National Institutes of Health guidelines (NIH Publications No. 8023). Patient samples used in this study were approved by the Human Research Ethics Committee of Huashan Hospital (No. 2017-041, approved 21 February 2017).

**Informed Consent Statement:** Each of the enrolled patients provided written informed consent.

**Data Availability Statement:** No additional data are available.

**Acknowledgments:** We are grateful to Yirui Li (School of Nursing, Fudan University) for her assistance in the correction of grammatical mistakes in this article.

**Conflicts of Interest:** The authors have no conflict of interests related to this publication.

#### **References**

## *Review* **Recent Progress in Lipid Nanoparticles for Cancer Theranostics: Opportunity and Challenges**

**Sarah I. Bukhari 1 , Syed Sarim Imam 1 , Mohammad Zaki Ahmad 2 , Parameswara Rao Vuddanda 3 , Sultan Alshehri 1,4 , Wael A. Mahdi 1 and Javed Ahmad 2,\***

**Abstract:** Cancer is one of the major leading causes of mortality in the world. The implication of nanotherapeutics in cancer has garnered splendid attention owing to their capability to efficiently address various difficulties associated with conventional drug delivery systems such as non-specific biodistribution, poor efficacy, and the possibility of occurrence of multi-drug resistance. Amongst a plethora of nanocarriers for drugs, this review emphasized lipidic nanocarrier systems for delivering anticancer therapeutics because of their biocompatibility, safety, high drug loading and capability to simultaneously carrying imaging agent and ligands as well. Furthermore, to date, the lack of interaction between diagnosis and treatment has hampered the efforts of the nanotherapeutic approach alone to deal with cancer effectively. Therefore, a novel paradigm with concomitant imaging (with contrasting agents), targeting (with biomarkers), and anticancer agent being delivered in one lipidic nanocarrier system (as cancer theranostics) seems to be very promising in overcoming various hurdles in effective cancer treatment. The major obstacles that are supposed to be addressed by employing lipidic theranostic nanomedicine include nanomedicine reach to tumor cells, drug internalization in cancer cells for therapeutic intervention, off-site drug distribution, and uptake via the host immune system. A comprehensive account of recent research updates in the field of lipidic nanocarrier loaded with therapeutic and diagnostic agents is covered in the present article. Nevertheless, there are notable hurdles in the clinical translation of the lipidic theranostic nanomedicines, which are also highlighted in the present review along with plausible countermeasures.

**Keywords:** cancer; multi-drug resistance; enhanced permeation and retention effect; nanotherapeutics; cancer theranostic; clinical translation

#### **1. Introduction**

At present, cancer is one of the leading causes of mortality worldwide. As per the demographic provided by the world health organization (WHO), cancer is accountable for approximately 10 million deaths in 2020 [1–4]. The emerging area of nanotechnology has proved very promising in cancer therapeutics [5]. Despite tremendous efforts in the research area of carcinomas alleviation through nanomedicines, there are a very few approved nanomedicines such as Doxil®, Myocet®, Abraxane®, Depocyt®, Genexol® [5,6]. The FDA-approved Doxil was thought to be a revolutionary lipidic nanomedicine when it was successfully developed. However, it was demonstrated in a clinical study that it exhibited poor therapeutic efficacy [7,8]. Harrington and colleagues conducted a study in

**Citation:** Bukhari, S.I.; Imam, S.S.; Ahmad, M.Z.; Vuddanda, P.R.; Alshehri, S.; Mahdi, W.A.; Ahmad, J. Recent Progress in Lipid Nanoparticles for Cancer Theranostics: Opportunity and Challenges. *Pharmaceutics* **2021**, *13*, 840. https://doi.org/10.3390/ pharmaceutics13060840

Academic Editor: Hassan Bousbaa

Received: 3 May 2021 Accepted: 2 June 2021 Published: 7 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

which they monitored tumor uptake of 111In-labelled poly(ethylene glycol) coated (PEGylated) liposomes in 17 patients with locally advanced cancers [9]. In this study, they were able to successfully demonstrate the major reason for the therapeutic failure of Doxil [9]. The study outcome revealed a very important and highly ignored aspect of in vivo fate of nanomedicines, targeting different forms of carcinomas. The study has confirmed that the accumulation of 111In-liposomes did take place in tumor restricted areas, but the concentration of nanoformulation that was retained in the tumor cells varied largely across different patients and tumor types [9,10]. In cancer patients, it has been established that enhanced permeation and retention effect (EPR) in which tumor vasculature becomes impaired and lymphatic drainage becomes deficient, felicitates drug accumulation in tumor cells, but that concept cannot be generalized after such findings [9,10]. Several factors affect nanomedicine accumulation and retention in tumor cells that need substantial consideration, such as tumor heterogeneity that causes diversified uptake of nanomedicines [11,12]. Tumor heterogeneity could be attributed to the distinctive cellular morphology, expression of efflux or influx transporters such as P-glycoprotein, presence of receptors, gene expression, metabolism, proliferation, and metastatic potential. fate of nanomedicines, targeting different forms of carcinomas. The study has confirmed that the accumulation of 111In-liposomes did take place in tumor restricted areas, but the concentration of nanoformulation that was retained in the tumor cells varied largely across different patients and tumor types [9,10]. In cancer patients, it has been established that enhanced permeation and retention effect (EPR) in which tumor vasculature becomes impaired and lymphatic drainage becomes deficient, felicitates drug accumulation in tumor cells, but that concept cannot be generalized after such findings [9,10]. Several factors affect nanomedicine accumulation and retention in tumor cells that need substantial consideration, such as tumor heterogeneity that causes diversified uptake of nanomedicines [11,12]. Tumor heterogeneity could be attributed to the distinctive cellular morphology, expression of efflux or influx transporters such as P-glycoprotein, presence of receptors, gene expression, metabolism, proliferation, and metastatic potential. Besides, there is considerable constraint at different levels of nanomedicine administration (as shown in Figure 1) journey in vivo that governs the overall therapeutic efficacy of nanomedicines. When nanomedicine is administered systemically, then it encounters different physical, chemical, and physiological barriers that hinder its reach to tumor

exhibited poor therapeutic efficacy [7,8]. Harrington and colleagues conducted a study in which they monitored tumor uptake of 111In-labelled poly(ethylene glycol) coated (PEGylated) liposomes in 17 patients with locally advanced cancers [9]. In this study, they were able to successfully demonstrate the major reason for the therapeutic failure of Doxil [9]. The study outcome revealed a very important and highly ignored aspect of in vivo

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 2 of 24

Besides, there is considerable constraint at different levels of nanomedicine administration (as shown in Figure 1) journey in vivo that governs the overall therapeutic efficacy of nanomedicines. When nanomedicine is administered systemically, then it encounters different physical, chemical, and physiological barriers that hinder its reach to tumor cells. After getting across these barriers, there are significant hindrances at the tumor microenvironment level that severely affect drug accumulation [11–13]. These hindrances include abnormal structure and highly variable density of tumor vasculature that greatly interfere with the optimal diffusion of nanomedicines. In addition, the pressure exerted by interstitial fluid and tightly packed tumor cells is very high, which avert the diffusion of nanomedicines across the tumor milieu. Moreover, a highly dense extracellular matrix greatly restricts nanoparticle extravasation and interstitial diffusion (Figure 1) [11–13]. cells. After getting across these barriers, there are significant hindrances at the tumor microenvironment level that severely affect drug accumulation [11–13]. These hindrances include abnormal structure and highly variable density of tumor vasculature that greatly interfere with the optimal diffusion of nanomedicines. In addition, the pressure exerted by interstitial fluid and tightly packed tumor cells is very high, which avert the diffusion of nanomedicines across the tumor milieu. Moreover, a highly dense extracellular matrix greatly restricts nanoparticle extravasation and interstitial diffusion (Figure 1) [11–13]. Therefore it was realized that there is a major lacuna in our knowledge of the pathophysiological complexities and heterogeneity of tumor sites that affect the therapeutic efficacy of nanomedicines. Those patients are not even identified who are likely to benefit most from given nanomedicine-based chemotherapy [14].

**Figure 1.** Barriers to efficacious in vivo performance of nanomedicines. **Figure 1.** Barriers to efficacious in vivo performance of nanomedicines.

Therefore it was realized that there is a major lacuna in our knowledge of the pathophysiological complexities and heterogeneity of tumor sites that affect the therapeutic efficacy of nanomedicines. Those patients are not even identified who are likely to benefit most from given nanomedicine-based chemotherapy [14].

Then next, the in vivo nanomedicine behavior knowledge is restricted to animal data and the animal models used do not mimic the actual in vivo conditions [15–20]. Usually, the NPs targeted for solid tumors after systemic administration are accumulated in the tumor through the EPR effect. Nevertheless, several crucial aspects related to EPR interpretation have been greatly overlooked, such as the influence of nanomedicine–protein interaction, blood circulation, tumor tissue penetration, and tumor cell internalization. Furthermore, all these biological processes are greatly affected by nanomedicine properties (for example, size, geometry, surface features) thus there are so many factors governing EPR effects driven in vivo nanomedicine behavior that cannot be predicted from animal data for humans. To date, there is not a single model that can completely replicate the entire facets of human malignancy [15–17]. This issue can be addressed if diagnosis and therapy can be combined as one approach in developing lipidic nanomedicines targeting cancers (Figure 2). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 4 of 24

**Figure 2.** Brief depiction of how lipidic theranostic can help in cancer alleviation via multi-functionalized aspects such as coupling of the imaging probe, surface with gold nanoparticles for photothermal therapy. **Figure 2.** Brief depiction of how lipidic theranostic can help in cancer alleviation via multi-functionalized aspects such as coupling of the imaging probe, surface with gold nanoparticles for photothermal therapy.

> **2. Significance of Lipid-Based Theranostic Nanoparticles in Cancer Therapy**  Early detection of carcinomas is of great pertinence for their successful alleviation. Firstly the diagnosis of the type of tumor, its metastatic state, and the patient history need to be identified to initiate the appropriate therapy. Here theranostic plays a crucial role. Once correctly and profoundly diagnosed, then the nanomedicine-based therapy could be started relevantly. However, the monitoring of the in vivo fate of nanomedicine is a vital aspect of assessing the progression and efficacy of cancer therapy. That is why coencapsulation of imaging agents and drugs in a single nanocarrier system could contribute significantly in assessing the progression of treatment and exact and precise state of the response of cancer towards provided therapy [21–23]. Theranostic nanomedicine could be prepared in multiple manners. The lipid nanocarriers have been proven superiority over polymeric and inorganic nanoparticles in terms of biocompatibility, safety, and biodegradability besides other beneficial consider-A cancer diagnosis has a very significant role in the context of precision of chemotherapeutic medication. Diagnosis specifically implicates recognizing the presence of a tumor in the body and evaluating its extent to identify if it is at its early developmental stage or re-occurrence case [1,2]. Importantly, identification of precancerous lesions could result in a successful intervention of cancer and its complete alleviation. Early diagnosis is certainly life-saving in cancer treatment. Once the existence of cancer is confirmed, diagnostic tools are implicated in identifying specific molecular abnormalities in tumors that govern the medications to be provided accordingly. Nowadays, with advances in biomedical technologies, novel diagnostic approaches are being investigated that will enable the identification of cancerous and precancerous cells at the molecular level and provide information about their pathophysiology. The combination of diagnostics with therapeutics makes it easy to know the progress of treatment and the real-time state of cancer while receiving therapy. Moreover, imaging agents/drug trackers can help greatly to know the in vivo fate or traveling of a drug in systemic circulation or at the tumor and can also determine the kinetics aspect of anticancer drug/drug loaded formulation [20,21]. That is why the use of imaging

> ations from a cancer theranostic perspective (as summarized in Figure 3) [7–9]. Therefore, in this review, we are concerned with lipidic theranostic for cancer, and we will be focus-

> nanoemulsions, the theranostic agent and drug are entrapped in oil globules targeted for specific tumor sites. In liposomes, imaging agents can be encapsulated with drugs either in an aqueous core or bilayer lipidic shell. In solid lipid nanostructure (SLN), the imaging agent is intercalated in the solidified lipid matrix. Whereas, in nanostructure lipid carrier (NLC), the drug and the imaging agent are dispersed in the imperfections of oil and solid lipid hybrid matrix. However, optimizing lipid theranostic nanomedicines with balanced size, shape, polydispersity index, surface charge, and stability, is a challenging task in

itself that we will discuss in detail in later sections.

biomarkers such as radioactive substances taken up by tumors and visualized through diagnostic modalities such as Computed Tomography (CT), Magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) is attaining a lot of attention in oncology these days. Taking into consideration the phenomenon of tumor metastases on the way to cancer mortality, the combinatorial approach of diagnosis and treatment that is theranostic will be of substantial importance for the assessment of EPR and nanomedicine penetration. The clinical translation of anticancer nanomedicines could see a breakthrough outcome via the introduction of a theranostic approach that can intently trace the in vivo fate of drugs and assess the progression of alleviation of human tumors via encapsulated nanomedicines [18–21].

Therefore, the diagnosis in combination with therapy is quintessential for embarking on a level of cancer treatment that could offer highly efficacious clinical outcomes. In this review, an insight of a combination of therapy and diagnosis, which is called theranostic, is provided, covering the brighter prospects and the challenges accompanied with it. The lipidic nanomedicine-based theranostic is also the highlight of this present review. A comprehensive account of different research updates in the field of lipidic nanocarrier loaded with diagnostic agents is envisaged. Nevertheless, there are notable hurdles in the clinical translation of the lipidic theranostic nanomedicines, which are also discussed in the upcoming section of the present review.

#### **2. Significance of Lipid-Based Theranostic Nanoparticles in Cancer Therapy**

Early detection of carcinomas is of great pertinence for their successful alleviation. Firstly the diagnosis of the type of tumor, its metastatic state, and the patient history need to be identified to initiate the appropriate therapy. Here theranostic plays a crucial role. Once correctly and profoundly diagnosed, then the nanomedicine-based therapy could be started relevantly. However, the monitoring of the in vivo fate of nanomedicine is a vital aspect of assessing the progression and efficacy of cancer therapy. That is why co-encapsulation of imaging agents and drugs in a single nanocarrier system could contribute significantly in assessing the progression of treatment and exact and precise state of the response of cancer towards provided therapy [21–23].

Theranostic nanomedicine could be prepared in multiple manners. The lipid nanocarriers have been proven superiority over polymeric and inorganic nanoparticles in terms of biocompatibility, safety, and biodegradability besides other beneficial considerations from a cancer theranostic perspective (as summarized in Figure 3) [7–9]. Therefore, in this review, we are concerned with lipidic theranostic for cancer, and we will be focusing on the vesicular or micellar lipidic structures. Different shapes and structures can be formulated depending on the type of lipidic nanocarrier chosen (as shown in Figure 4). In nanoemulsions, the theranostic agent and drug are entrapped in oil globules targeted for specific tumor sites. In liposomes, imaging agents can be encapsulated with drugs either in an aqueous core or bilayer lipidic shell. In solid lipid nanostructure (SLN), the imaging agent is intercalated in the solidified lipid matrix. Whereas, in nanostructure lipid carrier (NLC), the drug and the imaging agent are dispersed in the imperfections of oil and solid lipid hybrid matrix. However, optimizing lipid theranostic nanomedicines with balanced size, shape, polydispersity index, surface charge, and stability, is a challenging task in itself that we will discuss in detail in later sections.

The imaging agent that is incorporated with the drug in lipidic vesicles must possess great compatibility with conventional diagnostic techniques such as X-ray, ultrasound (US), CT, MRI, PET, or SPECT [24]. The contrasting agents used in theranostic usually include metals or inorganic agents (such as iron oxide) as they exhibit free electrons whose excitatory phase can be used as imaging modalities (MRI). Different semiconductor-based nanoparticulate systems (such as quantum dots) of colloidal dimension are also being extensively employed in in vivo diagnostics [25]. Fluorescent silicon nanoparticles are also employed as an imaging agent for foreseeing in vivo prospects [26].

MRI is one of the most commonly used diagnostic modalities, which depends upon mobile protons of water molecules for detecting signals. The MRI images can be perceived via proton concentration in body tissues or else via their longitudinal and transversal relaxation times, T1 and T2, respectively. A plethora of contrast agents are being used for MRI. Amongst them, fluorine-containing contrast agents have garnered splendid attention. Owing to its excellent magnetic property, 100% natural abundance, one-half spin, sensitivity, and gyromagnetic ratio comparable to a proton, fluorine has become the contrast agent of choice for MRI. Furthermore, with 19F in vivo tracing and imaging can be accomplished without any background signals created by endogenous fluorine. Nonetheless, there has been a limitation with fluorine as its large concentration (10–50 millimolar) is needed for adequate signal intensity in comparison to other contrast agents [27].

The promising combination of imaging along with nanomedicine-based therapy has the extremely worthy potential of overcoming the pathophysiological hurdles that undermine the efficiency of cancer therapy. The simultaneous examination of nanomedicine reach to tumor cells, amount of drug release, off-site tumor drug distribution, and uptake via host immune system are those aspects, which can tremendously affect the therapeutic outcome.

Until recently, the lack of interaction between diagnosis and treatment has hampered efforts to deal with cancer effectively. This new paradigm with simultaneous multifunctionality of imaging (with contrasting agents), targeting (with biomarkers), and delivering chemotherapeutic agents in one lipidic nanocarrier system seems to be very promising in overcoming various hurdles in effective cancer treatment. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 5 of 24

> **Liposomes:** Highly biocompatible, high drug loading, Long circulating half life via PEG coating, high accumulation in tumor tissue, diagnosting agent can br successfully incorporated, multifunctionalized capacity

**Nanoemulsion:** Effecient encapsulation and release of poorly soluble drugs, safe to administer, efficient accumulation in tumor cells, Biocompatible, nontoxic, multicomponent lodging

**SLN:** Sustained and substantialy increased accumulation of drug at tumor cells, prolonged in vivo circulation half life, non-toxic, biocompatible, theranostic agent can be easily encapsulated

**NLC:** Efficient encapsulation of poorly soluble drugs and theranostic agents, By pass hepatic uptake, promote lymphatic uptake, high drug loading capacity, sustained release of anticancer agents, enhanced antitumor activity

**Polymer-lipid hybrid:** High stability, greater drug loading, substantially enhanced anticancer and imaging potential, great scope of improvisation with different advancements in targeting strategies

**Figure 3.** Potential advantages of different types of lipidic formulations in cancer theranostic. **Figure 3.** Potential advantages of different types of lipidic formulations in cancer theranostic.

**Figure 4.** Different types of lipid nanoparticles *viz.* nanoemulsion, liposome, solid lipid nanoparticle (SLN), nanostruc-

tured lipid carrier (NLC), and micelles with significant utility in cancer imaging and therapy.

**Figure 3.** Potential advantages of different types of lipidic formulations in cancer theranostic.

**Liposomes:** Highly biocompatible, high drug loading, Long circulating half life via PEG coating, high accumulation in tumor tissue, diagnosting agent can br successfully

**Nanoemulsion:** Effecient encapsulation and release of poorly soluble drugs, safe to administer, efficient accumulation in tumor cells, Biocompatible, non-

**SLN:** Sustained and substantialy increased accumulation of drug at tumor cells, prolonged in vivo circulation half life, non-toxic, biocompatible,

**NLC:** Efficient encapsulation of poorly soluble drugs and theranostic agents, By pass hepatic uptake, promote lymphatic uptake, high drug loading capacity,

sustained release of anticancer agents, enhanced antitumor activity

**Polymer-lipid hybrid:** High stability, greater drug loading, substantially enhanced anticancer and imaging potential, great scope of improvisation with different

incorporated, multifunctionalized capacity

toxic, multicomponent lodging

advancements in targeting strategies

theranostic agent can be easily encapsulated

**Figure 4.** Different types of lipid nanoparticles *viz.* nanoemulsion, liposome, solid lipid nanoparticle (SLN), nanostructured lipid carrier (NLC), and micelles with significant utility in cancer imaging and therapy. **Figure 4.** Different types of lipid nanoparticles *viz.* nanoemulsion, liposome, solid lipid nanoparticle (SLN), nanostructured lipid carrier (NLC), and micelles with significant utility in cancer imaging and therapy.

#### **3. Different Types of Lipid Nanoparticles for Cancer Theranostics: An Update of Recent Studies**

Biocompatibility and safety are the major reasons for choosing a lipidic nanocarrier system as cancer theranostic in this review. Certainly, prolonged in vivo circulation halflife, high encapsulation capacities for drug and imaging agents, substantial accumulation at tumor sites, and improvisation susceptibility for multiple functioning are the other significant advantages with lipidic nanocarrier system [28–30]. Lipidic nanocarriers have their own sets of advantages, which give them an edge over other nanoformulations. Recent research studies demonstrated lipidic theranostic nanomedicines to be a promising and potential approach for raising the efficacy of cancer treatment to a hallmark level, as discussed hereunder. The different lipidic nanocarriers include liposomes, nanoemulsions, SLN, NLC, and lipid nanocapsules. However, advanced lipidic nanocarriers such as SLN and NLC are still unexplored for cancer theranostic as there are very few studies conducted thus far.

#### *3.1. Nanoemulsion*

Nanoemulsions have gained huge attention for the efficient delivery of lipophilic anticancer drugs. The nanometric size, large surface area, thermodynamic stability, high drug loading capacity, easy scalability, biocompatibility, favorable drug release profile are the characteristic attributes of nanoemulsion that make it worthy of profound exploration in chemotherapeutics [31,32]. The nanoemulsions, mostly oil-in-water types, are optically transparent colloidal dispersion constituted of oil and water, wherein surfactants and co-surfactants create a stable coating over the dispersed droplets to form a physicochemically stable nanoformulation [32]. The excipients of nanoemulsion are generally recognized as safe (GRAS) grade making the formulation highly safe to administer with improved bioavailability and therapeutic efficacy. Different strategies have been reported of late, wherein contrast and chemotherapeutic agents are entrapped in nanoemulsion for selectively targeting tumor microenvironment (TME) for both diagnostic and therapeutic drive [33–37]. Nonetheless, there are many obstacles in their journey from animal models to afflicted cancer patients, including firstly physicochemical stability of theranostic na-

noemulsion, subsequently, its in vivo fate, then degradation, and clearance from the system, along with long-term stability and toxicities concerns. Various attempts have been made thus far to surmount these hurdles and to entrap chemotherapeutic agents and diagnostic agents in nanoemulsion, particularly from the perspective of developing an efficient cancer theranostic agent that is highly capable of clinical translation (Figure 5). afflicted cancer patients, including firstly physicochemical stability of theranostic nanoemulsion, subsequently, its in vivo fate, then degradation, and clearance from the system, along with long-term stability and toxicities concerns. Various attempts have been made thus far to surmount these hurdles and to entrap chemotherapeutic agents and diagnostic agents in nanoemulsion, particularly from the perspective of developing an efficient cancer theranostic agent that is highly capable of clinical translation (Figure 5).

surfactants create a stable coating over the dispersed droplets to form a physicochemically stable nanoformulation [32]. The excipients of nanoemulsion are generally recognized as safe (GRAS) grade making the formulation highly safe to administer with improved bioavailability and therapeutic efficacy. Different strategies have been reported of late, wherein contrast and chemotherapeutic agents are entrapped in nanoemulsion for selectively targeting tumor microenvironment (TME) for both diagnostic and therapeutic drive [33–37]. Nonetheless, there are many obstacles in their journey from animal models to

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 7 of 24

**Figure 5.** Brief depiction of how lipidic theranostic can help in cancer alleviation via multi-functionalized aspects such as coupling of the surface with targeting ligand; entrapped theranostic agent, PEG coating on the surface for evading systemic clearance, bound oxygen for enhancing photothermal therapy, surface anchored contrasting agent. **Figure 5.** Brief depiction of how lipidic theranostic can help in cancer alleviation via multi-functionalized aspects such as coupling of the surface with targeting ligand; entrapped theranostic agent, PEG coating on the surface for evading systemic clearance, bound oxygen for enhancing photothermal therapy, surface anchored contrasting agent.

The major challenge associated with developing lipid theranostic nanomedicine, which is also the major limitation in their clinical translation, is that the incorporation of several components in one nanocarrier [38]. This integration of numerous constituents in nanodispersion causes polydispersity, heterogeneity, and also difficulty in scalability issues [38]. In an attempt to address these issues, Zhang and coworkers 2020, developed fluorinated nanoemulsions with remarkably improved fluorescence imaging signals for selective and sensitive tumor detection [38]. Their theranostic approach was highly capable of selectively recognizing the specific type of tumor (integrin αvβ3 overexpressed cancer cells), potential tracing of in vivo fate of nanoemulsion, and offering highly efficient photodynamic therapy. The meticulous approach averted many ingrained concerns with conventional nanomedicine, including polydisperse polymers, heterogeneous constituents, and complex formulation. Importantly, this approach imparted multiple functional aspects to the nanoparticles with tumor-targeting accompanied with quantitative and sensitive multimodal imaging (FL, 19F MRI, 129Xe hyper-CEST MRI), and PDT with a high therapeutic index [38]. Huang and associates 2019, designed an integrated system of mul-The major challenge associated with developing lipid theranostic nanomedicine, which is also the major limitation in their clinical translation, is that the incorporation of several components in one nanocarrier [38]. This integration of numerous constituents in nanodispersion causes polydispersity, heterogeneity, and also difficulty in scalability issues [38]. In an attempt to address these issues, Zhang and coworkers 2020, developed fluorinated nanoemulsions with remarkably improved fluorescence imaging signals for selective and sensitive tumor detection [38]. Their theranostic approach was highly capable of selectively recognizing the specific type of tumor (integrin αvβ3 overexpressed cancer cells), potential tracing of in vivo fate of nanoemulsion, and offering highly efficient photodynamic therapy. The meticulous approach averted many ingrained concerns with conventional nanomedicine, including polydisperse polymers, heterogeneous constituents, and complex formulation. Importantly, this approach imparted multiple functional aspects to the nanoparticles with tumor-targeting accompanied with quantitative and sensitive multimodal imaging (FL, 19F MRI, 129Xe hyper-CEST MRI), and PDT with a high therapeutic index [38]. Huang and associates 2019, designed an integrated system of multimodal imaging signals and PDT function into a poly(ethylene glycol)-boron dipyrromethene amphiphile (PEG-F54-BODIPY) with 54 fluorine-19 (19F), as an "all-in-one" nanocarrier [39]. This novel amphiphile acquired various unique and desirable attributes that make it potential cancer theranostic agents. The developed nanoemulsion was having distinctive structure-based fluorescent, photoacoustic, and magnetic resonance properties and prolonged tumor retention time for repeated PDT treatment, and great biocompatibility. The study outcomes revealed in the melanoma cancer xenograft model, developed nanoemulsion can be efficiently used as an activatable nanoprobe with improved sensitivity of multimodal imaging

for tumor recognition [39]. One more very interesting attempt was made by Fernandes and Kolios 2019, to increase the selectivity and targeting of nanomedicine for substantially afflicting cancer cells without causing any harm to nearby cells and off-tumor areas [40]. The fabricated perfluorohexane nanoemulsions possess the ability to selectively target cancer cells as these nanoparticles carry therapeutic agents, which have slow release rates and become vaporized into perfluorohexane bubbles without any rise in temperatures that could affect normal cellular function. Their ability to use higher concentrations of theranostic agents could be of significant advantage in improving therapeutic efficacy and imaging ability in clinical applications. In this study, the ability of nanoemulsions to cart therapeutic agents, doxorubicin, and paclitaxel, specifically for the treatment of breast cancer, was investigated. The study outcome revealed that perfluorohexane nanoemulsion could be efficiently internalized in cancer cells and could cause significant cell death. The developed nanoemulsion with concurrent laser excitation capability exhibited tremendous potential for destroying all tumor cells and emerging as a competent theranostic agent for averting the growth of cancer cells [40]. Furthermore, Zheng and associates, 2019 fabricated a novel nanoemulsion with a porphyrin shell (NewPS), and it was the simplest yet multifunctional nanoemulsion system developed thus far [41]. The porphyrin salt shell permitted the encapsulation and stabilization of the oil core, yielding a monodisperse, spherical nanostructure with excellent colloidal stability. The inherent multimodality of porphyrins enabled the multifunctionality of NewPS for imaging and phototherapy. Moreover, the oily core felicitates the efficient loading of hydrophobic anticancer molecules. The study established formable and intelligible, surfactant-free nanoplatforms for theranostic cancer applications. This novel two-component NewPS served as an innovative avenue for multimodal cancer imaging, phototherapy, and image-guided drug delivery [41].

In light of such studies, it is anticipated that nanoemulsions-based theranostic could offer promising opportunities in cancer treatment.

#### *3.2. Liposomes*

Liposomes stand tall in the crowd of conventional lipidic nanocarrier systems owing to their inimitable characteristic attributes. Their unique structure comprised of unilamellar lipid bilayers that nest an aqueous core offers great flexibility of easy incorporation of multicomponent. It also provides an option for both hydrophilic and lipophilic chemotherapeutic drugs and contrast agents. Besides biocompatibility, safety, biodegradability aspects, liposomes also offer an enormous scope of surface improvisation for selectively targeting tumor cells [42]. Recent literature highlights that liposomes have been amongst the topmost area of active research of cancer theranostics. At present, they are being largely investigated for incorporating and targeting cancer via contrast agent such as 64Cu [43] and 14C isotopes [44], QDs [45], gadolinium (Gd)-based contrast agents [46], super paramagnetic iron oxide particles (SPIONs) [47], and fluorescent probes [46,48].

The upcoming section will throw light upon the latest research studies that have confirmed the substantial potential of liposomes as cancer theranostic that could be successfully taken to clinics.

Prasad and associates 2021; fabricated liposomal nanotheranostics in which gold nanoparticles (AuNPs) and emissive graphene quantum dots (GQDs) were encapsulated along with a chemotherapeutic agent [49]. The surface of the liposome was functionalized with folic acid for targeted delivery. The prepared targeted liposomal theranostic demonstrated site-specific tumor diagnosis and photo-triggered tumor extirpation. The study outcome confirmed specific and enhanced cellular uptake, prolonged internalization in tumors, and substantial contrasting and therapeutic efficacy of nanotheranostic liposomes with dual imaging probes. The synergistic effect of anticancer drugs and photothermal effect exhibited superior tumor impedance, in contrast, to stand-alone therapy. Moreover, these multicomponent loaded liposomes have good colloidal stability, biocompatibility, and biodegradability as demonstrated in in vivo imaging. Therefore the developed nano

hybrid liposome nanotheranostic served as a safe and tremendously potential platform for multifunctional tumor diagnosis and targeted therapy [49].

Furthermore, Karpuz and associates 2020, investigated the in vivo therapeutic prospect and contrasting efficacy of paclitaxel and vinorelbine loaded, Tc-99m radiolabeled, folate targeted nanosized liposomes [50]. The study outcomes demonstrated targeted delivery of chemotherapeutic agents, which got efficiently lodged in tumor vasculature and resided there for a prolonged time, causing substantial reduction of tumor growth. The in vivo images confirmed mitigated off-site accumulation and toxic effect of liposome theranostic nanomedicines [50].

In another interesting research study, a very serious issue of brain metastasis (BM) and tyrosine kinase inhibitor (TKI) resistance that are the two major challenges in non-small cell lung cancer (NSCLC) treatment were addressed [51]. Yin and associates designed a dual-targeting liposomal system for co-delivery of gefitinib and simvastatin to treat BM of NSCLC. The study confirmed via fluorescence imaging that dual-targeted liposome could efficiently cross the blood–brain barrier and is highly capable of reversing drug resistance. Therefore, the developed liposomal formulation represents a potential strategy for treating advanced NSCLC patients with BM [51].

Bush and coworkers 2020 also came up with an interesting concept of acoustic cluster therapy (ACT) that constitutes of co-administration of a formulation containing microbubble constellations, along with regular anticancer drug and local US insonation of the targeted tumor tissue. The microbubble cluster efficiently resided in the tumor's microvasculature [52]. The therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple-negative breast cancer using orthotopic human tumor xenografts in athymic mice was assessed. The study outcome established substantial increase in the therapeutic efficacy of Doxil® when combined with ACT [52]. Another crucial study that encourages the concept of application of liposome in cancer theranostic was conducted by Prabhakar and Banerjee 2019 [53]. They formulated submicron-sized (528.7 ± 31.7 nm) nanobubble-paclitaxel liposome (NB-PTXLp) complexes for ultrasound imaging and ultrasound responsive drug delivery in cancer cells. The concept resulted in more than 300-fold higher anticancer activity of NB-PTXLps in the presence of ultrasound in MiaPaCa-2, Panc-1, MDA-MB-231, and AW-8507 cell lines, in contrast to commercial formulation Abraxane®. Therefore, the novel NB-PTXLps served to be a promising and triflingly invasive theranostic scaffold for cancer therapy in the forthcoming scenario [53].

The research studies discussed in this section undoubtedly unveiled enormous opportunities to facilitate the targeted chemotherapeutic delivery with concomitant in vivo imaging utilizing liposomes. Further research is envisaged to take these studies to clinical trials.

#### *3.3. Solid Lipid Nanoparticles (SLN)*

The second-generation lipid nanocarrier includes SLN, which are spherical colloidal nanoparticles with a solid lipid core comprised of waxes, triglycerides, fatty acids, and are stabilized by surfactants. Their size usually falls within the 50–100 nm range and is exclusively known for their biocompatibility, higher susceptibility of lymphatic uptake, and sustained drug release [54,55]. In cancer, alleviation chemotherapeutics loaded SLN is very promising [54–56]. Nonetheless, they are capable of efficiently carrying contrast agents along with anticancer drugs and provide simultaneous treatment and diagnosis, as evident in outcomes of recent research studies. Kuang and coworkers have demonstrated in their study that solid lipid nanoparticles (SLNs) conjugated with c(RGDyK) were designed as efficient carriers to improve the targeted delivery of IR-780 to the tumors [57]. The multifunctional cRGD-IR-780 SLN significantly improved the tumor-specific targeting, efficacy of photothermal therapy along with spontaneous imaging of in vivo journey of SLN incorporated IR-780 iodide nanomedicine [57].

SLNs have been investigated for integrating many contrasting agents, particularly superparamagnetic iron oxide [58], technetium-99 (99mTc), 64Cu [59], and quantum dots [60,61]. Very recently, a research study came up with SLN cancer theranostic wherein SLN was

encapsulated with QD as a contrast agent [54,62]. The chemotherapeutic agent was the combination of Paclitaxel and siRNA. The solid lipid matrix was interspersed with paclitaxel and QD whereas siRNA, which was anionic, was electrostatically linked on the outer cationic surface. The combination of dual therapeutic agent paclitaxel and siRNA loaded in SLN got efficiently accumulated in lung carcinoma and exhibited synergistic anticancer activity. Importantly QD fluorescence in SLN made it possible to track higher in vivo cellular uptake of SLN on-site and mitigated uptake at off-site cancer cells. This study confirmed the potential theranostic applicability of SLN as a nanocarrier [54,62].

In another interesting research study, Morel and associates have revealed the successful integration of gadolinium (Gd) (III) complexes in SLN that would be contributing as an efficient and favorable oral contrast agent for MRI [63]. The percentage of Gd (III) complex entrapped in SLN was substantially higher as confirmed by average longitudinal relaxivity for Gd (III) complex in SLN system and pure water (25 ◦C, pH 7, 20 MHz) [63].

Another research study conducted by Andreozzi and associates is clear evidence of the multifunctional theranostic ability of SLN [59]. They radiolabelled SLN with 64Cu and assessed its bio-distribution by in vivo quantitative assessment, PET imaging, and ex vivo gamma counting. The study outcomes validated the simultaneous in vivo imaging and tumor ablation potential SLN theranostic, which is also safe, biocompatible, and biodegradable [59].

The research studies conducted thus far are very limited in numbers, and several other important aspects of SLN also need profound exploration from a cancer theranostic perspective, such as stimulation of lymphatic absorption by SLN. Whatever literature we have discussed can corroborate the efficient in vivo imaging and drug delivery utilizing SLNs along with the safe theranostic application, biocompatibility, and biodegradability, of developed nanomedicine. The results established that the SLN theranostic nanoformulation developed is optimistic for hallmark contribution in the field of cancer treatment with simultaneous diagnosis.

#### *3.4. Nanostructured Lipid Carriers (NLC)*

NLC is a smart next-generation nanocarrier with a unique hybrid matrix of solid and liquid lipids stabilized by surfactants. The imperfect crystalline or amorphous structure possesses an enormous potential of high drug loading and improvised drug release. Latest research studies indicate the promising role of NLC in cancer theranostic. Of late, Li and coworkers successfully developed a multifunctional nanocarrier of Coumarin 6 fluorescent dye and IR 780 encapsulated CXCR4-targeted NLCs for breast cancer alleviation also employing photodynamic therapy [64]. The developed system proved to be highly efficient in debilitating tumor progression and metastasis and concurrently allowing imaging [64].

Olerile and coworkers prepared a NLC loaded with QD and paclitaxel that was highly capable of monitoring and tracking tumor growth and simultaneously impeding tumor cells in the murine tumor model of hepatocellular carcinoma [65]. Researchers confirmed the great cancer theranostic potential of NLC as it was efficiently enabling targeted delivery with concomitant in vivo imaging [65].

Another very promising approach in the area of cancer theranostic utilizing NLC was reported. Camptothecin encapsulated-NLC was formulated conjoined with QD and fluorescent imaging probes that were capable of detecting the lodging, internalization, cytotoxicity, and biodistribution of NLC nanomedicine [66]. The study outcome established that NLC coordinated with QDs and an anticancer drug offered efficient tumor imaging and drug delivery and such accomplishment with a novel nanocarrier system was remarkable and worth mentioning here [66].

An interesting attempt was made to deliver paclitaxel-loaded NLC with folic acid dispersed hybrid lipid matrix [67]. The paclitaxel-loaded NLC was radiolabeled with 99mTc(CO)3+. Due to the imperfection in a matrix structure, too many components were efficiently loaded in NLC as reflected by in vitro stability of developed formulation, which was up to the mark (>90%). Results indicated that 99mTc(CO)3+-radiolabelled paclitaxel NLC

was capable of identifying folate receptors overexpressed in tumor cells. The developed formulation was successfully localized at the specific targeted tumor areas without any off-site distribution and the uptake by RES on intravenous administration. The concurrent imaging efficiently depicted the in vivo fate of paclitaxel-loaded NLC that is very much desirable for an optimal therapeutic implication [67].

As the data concerning NLC theranostic for cancer is very restricted, there is a great need for a lot more investigation to be envisaged to explicitly explore multiple beneficial aspects of advanced lipid nanocarrier and pave the way for their successful clinical translation.

#### *3.5. Lipid Nanocapsules (LNCs)*

LNCs are also amongst the next-generation lipid nanocarrier systems with lipoproteinlike structures whose size falls within 1–100 nm. The structure of LNCs is a blend amidst polymeric nanoparticles and liposomes as they have an oily core with an exterior built of a tensioactive rigid membrane. LNCs are a novel lipid nanocarrier system and can be prepared via phase inversion of emulsions and organic solvent-free-based procedures [68].

Nevertheless, researchers consider LNCs as an optimistic platform for cancer theranostic as well. A very recent study further confirmed the promising outlook of LNCs in cancer theranostic. To selectively target tumor cells, QDs-based lipid nanocapsules (LNCs) encapsulated with celecoxib, and honokiol were designed and investigated. The study outcome revealed efficient accumulation and intracellular uptake of LNCs in tumor cells, and their internalization was progressively traceable via fluorescence restoration. The LNCs established highly improved and superior anticancer efficacy of LNCs against breast cancer cells. The developed system could be applied as a potential multifunctional nanotheransotic for imaging and targeted therapy of breast cancer [69].

#### *3.6. Lipid-Based Micelles*

Lipid-based micelles are the spherical structure of lipid molecules, in which they aligned themselves in aqueous solutions. This class of lipid nanocarrier system is unexplored to date, but it could also serve as a potential and promising therapeutic cum diagnostic nanomedicine for cancer treatment. In a study, Ma and coworkers developed a lipid-based micelle encapsulated with docetaxel (M-DOC) that possessed marked anticancer efficacy and mitigated toxicity in the xenograft breast cancer model [70]. The lipid-based micelles need to be duly explored for their cancer theranostic potential in the near future.

Table 1 enlists the different lipidic nanomedicine investigated for the theranostic applications in cancer and summarizes their theranostic outcomes in different experimental models.

#### **Table 1.** *Cont.*

#### **4. Advancement in Lipid-Based Nanoparticles for Cancer Theranostics**

*4.1. Polymer-Lipid Hybrid System*

Lipid polymer hybrid is next-generation lipid nanocarriers and has obtained its foundation from the amalgamation of liposomes and polymeric nanoparticles. They have a polymeric core enclosed by a lipid bilayer shell kind of structure. Many researchers claim it to be a very promising nanocarrier for anticancer drug delivery, however, its potential is not duly tapped and remains unexplored to date. In recent research, Huang and associates developed multifunctional tumor-targeted polymer-lipid hybrid nanoformulation, which was loaded with ultrasound contrast agents (glutathione (GSH)) and prodrug (Pt(IV)) for targeted delivery of theranostic nanomedicine against ovarian cancer. The nanosized formulation was composed of a perfluorohexane (PFH) liquid core, a hybrid lipid-polymer shell, and an active targeting ligand, which demonstrated improved cellular uptake. The study findings established Pt(IV) encapsulated lipid-polymer as a novel multimodality

platform exhibiting excellent anticancer activity and substantially reduced toxicity and signifying a powerful theranostic nanomedicine for combating ovarian cancer [71]. The research outcome encourages further exploration of this class of lipidic nanocarriers to be envisaged for beneficial prospects in the field of cancer theranostic.

#### *4.2.  HDL-like peptide- phospholipid nanovesicles (HPPS) imitate the structural and functional attributes of plasma-derived HDL [74–77]. He and coworkers recognized a TfR mAb (monoclonal antibody) tailored nanomedicines for improved tumor targeting. They demonstrated that drug entrapped HPPS based nanomedicines adapted with explicit ligands could muddle to receptors on the surface of tumor cells, resulting in the accretion of nanomedicines on the exterior surface of targeted cells [75–90]. It was concluded that developed HPPS based nanomedicine holds the potential to strengthen targeting to tumor cells and attains favored recognition by specific antibodies in a complex biological setting [91]. Such intricately woven novel research concept needs to be brought forth and encouraged thus that these kinds of potential research perceptions can be multiplied. Huge benefits can be extracted from such studies for accomplishing successful targeting of lipidic cancer theranostic nanomedicine.

Fayad and coworkers presented very interesting work on HDL-based multimodal nanotheranostic for targeting and imaging tumors [92]. The developed HDL nanoparticles got non-selectively accumulated and selectively binding to angiogenic active blood vessels. For targeting such angiogenic endothelial cells, HDL was reconstituted with gadolinium chelates and fluorescent dyes, and their surface was functionalized with αvβ3-integrinspecific RGD peptides. The incorporation of such peptides felicitated uptake of HDL-based nanoparticles by angiogenic endothelial cells as visualized in MRI after administration of developed nanoparticles in tumor-bearing mice. The study findings demonstrated the substantial possibility of redirecting HDL from their natural route towards tumorridden blood vessels along with successful imaging and tracing of an entire pathological process [92].

#### *4.3. Hybrid Lipid-Inorganic Nanomaterials*

Most recently, there has been a paramount focus shifted towards an exploration of hybrid lipid-inorganic nanomaterials, which combine and multiply the desirable attributes of both classes of nanocarriers, including lipidic nanocarriers and inorganic nanoparticles. The lipid nanocarrier system employed for such application includes liposomes, nanoemulsion, solid lipid nanoparticles, and lipoproteins. In contrast to singly functionalized counterparts, this hybrid multifunctional system offers many beneficial outcomes in cancer theranostic such as stimuli-triggered anticancer drug release, photothermal therapy, and bioimaging. The internalization of inorganic material inside the lipid nanocarrier governs their functional aspects as there are different spatial lodging based on the structure of lipidic assemblies. The inorganic material can reside in the surface coating of lipid nanocarriers as surface-bound nanomaterial, or it can be lodged in bilayer lipids lamellae in liposomes, and certainly, the inorganic material can also be internalized in the core structure of lipidic nanocarrier [93]. A plethora of investigational studies are reported in which gold (Au) was used as an inorganic nanomaterial for preparing hybrid lipid-inorganic nanoparticles for cancer theranostic. The considerably low toxicity, ease of improvising surface chemistry, tunable size and shape, and substantial electronic property make Au a metal of choice for preparing inorganic nanoparticles and their hybrid lipid nanoformulations that have great potential for bioimaging site-specific drug release, and photothermal cancer therapy [93]. Other potential inorganic materials that have been explored for preparing hybrid lipidinorganic nanomaterial include silver and palladium nanoparticles. In addition, one of

the most commonly used approaches include SPIONs, which are ideal contrast agent for MRI owing to their biocompatibility and distinctive magnetic properties [94]. Moreover, the potential of semiconducting nanoparticles (QDs) is becoming widely accredited in bioimaging as optical probes over conventional organic dyes [95].

The recent study findings focusing on hybrid lipid-inorganic nanomaterials are clear evidence of their potential in cancer theranostic. In a study, Kang and Ko have developed a hybrid lipid inorganic nanomaterial by efficiently incorporating docetaxel in Au nanoparticles and then encapsulating this Au nanoparticle in thermosensitive phospholipid lipid bilayer coating [96]. The outcome of the study established enhanced cellular uptake, internalization, and cytotoxicity of hybrid lipid inorganic nanoformulation in comparison to uncoated drug-loaded Au nanoparticles. The study findings strongly encourage the implication of drug-encapsulated lipid-coated anisotropic nanoparticles for amplifying therapeutic prospects of chemotherapy [96]. The recent research concluded the feasibility of breast cancer cell detection via conformance of the inorganic metal-nanoemulsion hybrid [97]. An improvisation was made in perfluorocarbon or QD nanoemulsions by incorporating N-hydroxysuccinimide modified phospholipids in the surfactant formulation, as this would enable conjugation of prepared hybrid QD nanoemulsion with the amine groups in antibodies. Such antibodies targeting growth factors are overexpressed in human breast cancer cells, which would be easily able to bind with nanoemulsions. The research study demonstrated the selective linking of hybrid nanoemulsion with its target breast cancer cell line [97]. Interestingly, low-density lipoprotein (LDL) encapsulating Au nanoparticles were explored for their biolabeling capability [98]. Administration of dodecanethiolcapped Au nanoparticles loaded LDL in mice with B16-F10 tumor resulted in selective uptake by tumor-associated macrophages that play a vital role in metastasis of tumor cells. The study findings confirmed the substantial potential of hybrid Au-LDL nanoformulation in in vivo tracking and treating of tumors without causing off-site damage [98]. In another study by Bao and coworkers, hybrid liposome nanoformulation wherein paclitaxel Au nanoparticles were embedded in its bilayer lipid lamellae demonstrated remarkably prolonged release rate and extended circulation time. The hybrid exhibited notable potential for enhancing the therapeutic efficacy of incorporated anticancer agents [99]. Mounting evidence has elucidated the potential of palladium nanoparticles as contrast agents for photothermal and anticancer therapy. Nevertheless, research works focused on hybrid lipid assemblies incorporating such palladium nanoparticles established excellent anticancer activity as well as site-specific uptake and internalization of a therapeutic agent through these hybrids [100,101]

Certainly, there are serious toxicity concerns with the use of inorganic nanomaterial. For example, oxidation of silver (Ag) to toxic Ag+ ion in biological milieu can cause toxic effects that must be controlled. Here hybrid lipid encapsulation could come to the rescue of such lethal transformation. By tuning the lipidic membrane composition, the release of Ag+ ions can be prevented. Moreover, studies have established that the physicochemical features, size, shape, surface, coating, surface area contribute significantly in dictating the hybrid lipid inorganic nanomaterial toxicity and its biological interactions [102]. Therefore, skillfully designed hybrid lipid inorganic nanomaterial exhibits the potential of overcoming the toxicity issue of incorporated metals.

#### *4.4. Cancer Tumor Cell Targeting Theranostic Vector*

At present, the active area of targeting circulating tumor cells (CTC) via theranostic vectors before the homing and progression of carcinomas is largely explored for improving cancer therapeutic intervention. The CTCs are those cells that are detached from primary solid tumors and traverse through blood and lymph to form secondary tumors [103]. Therefore, detection and targeting of CTC may result in early diagnosis and prevention of cancer and its metastasis. Determination of CTC concentration in blood could provide valuable information about the diseased state [103]. Therefore, estimating CTC concentration would contribute significantly in monitoring remission and relapse and assessing response to

chemotherapy [103]. In a study by Bhattacharyya and associates, antibody-targeted Au nanoparticles were employed for CTC detection in breast cancer cell line T47D [103]. The study outcomes established Au nanoparticles as a promising approach for detecting and capturing CTC in a photoacoustic flowmeter. The study findings established an abundant scope of diagnosis of disease at an early stage in various solid tumors and its successful treatment. Such encouraging findings mark a new paradigm for cell-specific molecular analysis for individualized cancer therapy via capturing of CTC [103].

#### **5. Impact of Physicochemical Attributes of Lipid Nanoparticles in Improving In Vivo Performance of Cancer Theranostics**

A recent study conducted by Tahmasbi and associates established that physicochemical facade, predominantly shape and size, greatly influences the efficiency of the lipidic theranostic nanomedicines [104]. Mounting literature confirmed that spherical nanomedicines having a diameter within 20−100 nm lead to optimal tumor accretion owing to the EPR effect. However, dissimilar EPR attributes have been testified owing to nonspherical nanostructures (i.e., nanorods) [104,105].

The EPR effect refers to the selective buildup of lipidic theranostic nanomedicine at the tumor locations through extravasation via endothelial cells from the dripping vasculature. Accounting to the recent evidence, the physicochemical attributes of the lipidic theranostic nanomedicine comprising size, shape, charge, etc., possess greater potential in dictating tumor accumulation in contrast to active targeting on the exterior of nanomedicines [105]. Consequently, lipidic theranostic nanomedicine can be formulated for targeted tissues as well as for non-specific cell absorption by optimizing their physicochemical properties in the absence of targeted ligands [105]. Lately, it has been verified that shape greatly impacts cellular uptake [106,107]. Myriads of studies confirmed the diversified morphology-dependent anticancer efficacies for the same chemical compositions [108,109]. For the discoidal shape of lipidic nanomedicine having sizes <50 nm, tumor accumulation efficiency is not explored until recently [110–113]. For lipidic theranostic nanomedicine greater than 100 nm, needle-like rods have shown the maximum cellular uptake, trailed by shapes such as spheres, cylinders, and cubes, as evident in supporting studies [104,105]. For sub-100 nm lipidic theranostic nanomedicine, spheres demonstrated enhanced uptake by tumor cells over rods [104,105].

Nonetheless, the fluidity of lipid membranes can fluctuate with charge (negative or positive) and may persuade local gelation [104]. Neutral and slightly negative lipidic theranostic nanomedicine are found to maintain the longest half-life in blood [104,112,113]. Lipidic theranostic nanomedicine that has a positive charge could ensue the issues of platelet aggregation and hemolysis [104,112,113]. The competence of such a platform to fine-tune surface charge is another benefit in addition to size and shape adjustment that plays a crucial role in improvising biological behavior and clinical outcomes of lipid theranostic nanomedicines [104].

The absence of specificity with inactive targeting determined by the nanosize of lipidic theranostic nanomedicine has restricted efficiency. Mounting literature revealed that active targeting to the tumors might augment the intracellular uptake and lessen the lipidic theranostic nanomedicine's spread in healthy tissues. A rational approach to attain this objective is to exploit specific interactions between the targeting molecules on the lipidic theranostic nanomedicine's surface and overexpressed receptors of the cancer cells. Few ligands, namely transferrin, and folate can considerably enhance site-specific targeting [104]. Predominantly, folate has emerged as one of the targeting ligands for selective delivery of involved diagnostic and therapeutic agents owing to the overexpressed folate receptor (FR) in a myriad of tumor tissues, including kidney, lung, ovarian, cervical, breast, epithelial, brain and colon tumors, whereas limited in healthy organs and tissues. Folic acid being nonimmunogenic, unchanging over wide ranges of temperatures and pH values, can bind to the folate receptor after pairing with drugs or imaging markers. Consequently, folic acid has been extensively used as targeted therapy in cancers on account of its high binding

affinity to FR, simplicity of conjugation to different lipidic theranostic nanomedicine, and the widespread distribution of FR in a substantial portion of human cancers [104]

#### **6. Limitation of Lipid Nanoparticles-Based Cancer Theranostics**

The major limitation with lipidic nanoparticles is their tendency to fuse, especially if the size of prepared nanoformulation is below 100 nm [114]. The fusion, in turn, results in increased non-uniformity and dispersity in size and escape of encapsulated contents from the lipid vesicles [114]. However, this issue can be addressed by covering the surface of lipid nanoparticles with polyethylene glycol coating. Another issue of great concern with lipidic nanoparticles is their stability over a longer duration of periods. It is certain that after storage for long periods, apparently intact lipid nanomaterials show a different biodistribution due to changes in physicochemical characteristics and alterations of the surface coating attributes [114].

Nonetheless, Carregal-Romero and associates have raised forth this very significant aspect of the influence of long-term stability of polyethylene glycol coating on contrast agent encapsulated nanoparticles and its in vivo fate [114]. They investigated the biodistribution of iron oxide nanoparticles, which were employed as dual contrast agents for MRI and SPECT imaging [114,115]. They developed these theranostic nanoparticles by coprecipitation of 111In-doped magnetic nanoparticles, followed by coating with polyethylene glycol [115]. Then they examined the physicochemical characteristics of freshly prepared nanoparticle solution and an aged nanoprobe solution that was stored for 9 months. The characteristic evaluation demonstrated comparable results of size distributions, zeta potentials, and morphology. However, after systemic administration of these two nanoparticle preparation in mice, completely distinct biodistribution pattern were observed [115]. The freshly prepared nanoprobe solution was mostly internalized in the kidney, whereas the aged nanoparticles were heavily concentrated in the liver. Therefore, the study outcomes concluded that there might occur some small level structural changes in polyethylene glycol coating that cannot be detected by dynamic light scattering and transmission electron microscopy, which have led to a remarkable alteration in in vivo behavior. Therefore this report established a substantial role of long-term stability polyethylene coating in dictating the biological behavior of theranostic nanoparticles [115].

The size of theranostic nanomedicine is also very crucial in determining its in vivo fate and clinical outcomes. It has been established in different studies that if the diameter of nanoparticles is larger than 100 nm then the particles get accumulated in the liver and spleen, whereas nanoparticles having a diameter smaller than 10–15 nm are usually eliminated by renal clearance [114]. Therefore nanomedicines with desirable particle diameters between 10 and 100 nm are supposed to have longer blood circulation times and accessibility to tumoral tissues and organs [114]. Furthermore, low polydispersity index (PDI) and size stability are very significant as they avert aggregation of nanoparticles, which could occur because of an inappropriate surface coating. The aggregated nanoparticle can be easily engulfed by the RES [114,115]. Nonetheless, polyethylene glycol coating is very effective in ensuring stability to the nanoparticles and thereby mitigating their opsonization, macrophage uptake, and RES clearance and increasing the blood circulation time [114].

Importantly, with every approved lipid nanomedicine, a possibility of intratumoral heterogeneity and variability of response to chemotherapy is evident. Unquestionably, the tumor microenvironment contributes largely in dictating how chemotherapeutic agents interact with cancer cells in that particular microenvironment, which in turn can impact proliferation, differentiation, morphology, and a range of cellular functions [116]. To envisage the clinical outcome of lipid-based theranostic nanomedicine, substantial emphasis should be given to universal heterogeneity issues with extraordinary conformity in terms of cancer cells, tumor microenvironment, and pathophysiological architecture [116]. However, with current theranostic and imaging modalities, complete biological approximation of the

interaction of native tumor and a chemotherapeutic agent is not possible, which greatly hampers the accurate mapping and clinical findings.

#### **7. Challenges in Clinical Translation of Lipid Nanoparticles for Cancer Theranostics**

An extensive scope has been conveyed by lipidic theranostic nanomedicine in enhancing the health of humans through providing an understanding of diagnosis, prevention, and treatment of diseases. Even after substantial technological advancement in this area (as presented in Table 2), there is still a long way ahead for lipidic cancer nanotheransotic in becoming a new criterion for the diagnosis and treatment of diseases. Nano-bio interaction is a prime opposition for the transferal of lipidic theranostic nanomedicine to clinics. Disorders like inflammation, immunoreactions, or related illness can come up when a contrasting agent in lipidic theranostic nanomedicine interacts with biological material because of its incompatibility or potential toxicity. The extent of toxicity is immensely based on some parameters, which include the solubility of the nanoformulation, size, and zeta-potential [117]. Once entered into a biological system, nanovesicles tend to interact with proteins, and that results in the development of 'corona' on its surface. Such adsorption, in turn, results in the alteration of their stability, biodistribution, dispersibility, toxicity profile, pharmacokinetics, and size [118,119]. This has also been shown in many studies that adverse immunogenic reactions and allergies are happening by nanoparticles [120,121]. Hence, it becomes vital to study the physicochemical characteristics of nanomedicines for heterogeneity and pathophysiology of human diseases. More importantly, a generalized size outlook of lipidic theranostic nanomedicine is not possible as chemotherapy is distinct for every patient, and this may pose a hurdle in clinical translation [122]. Nanomedicines with good therapeutic efficacy might not be a good diagnostic tool necessarily, as suggested by the evidence. In a recent study, it was found that tumor uptake and tumor visualization performed with anti-EGFR coated gold nanoparticles with 20 nm size showed increased tumor uptake, whereas the same gold nanoparticles of 50 nm size illustrated excellent CT contrast [123]. This study suggested that the size-dependent distribution of theranostic nanomedicines in tumors limits its use as a theranostic agent [123]. Hence, the safety profile of nanotheransotic in humans continues to be a major concern for which long-term monitoring of patients in both early and advanced phases of clinical trials is required. One more important obstruction for the clinical translation of lipidic theranostic nanomedicines is the complexity in formulating a reproducible and controllable synthesis process. Nanomedicines synthesis on a large scale faces challenges like varied physical and chemical characteristics, low yield, and insufficient batch-to-batch reproducibility. The complex and laborious manufacturing process of nanomedicines makes it difficult to focus on physic-chemical attributes as the emphasis is more on quality and cost [122,124].  Moreover, control along with good manufacturing practices are required to boost lipid theranostic medicine's reach to clinics from a laboratory.

**Table 2.** Lipidic nanocarrier based cancer theranostic in clinical stage of progress.

Another important issue that is required to be addressed is the extensive gap between regulatory authorities and the scientific community. Many government agencies are monitoring the commercialization of nanomedicine based on regulatory issues related to the safety profile, quality control, patent protection, and manufacturing practices. Lack of clear regulatory and safety guidelines affects the timely and effective translation of lipidic theranostic to market [125,126]. Even though the general regulatory standards have been cleared by the nanomedicines presently available in the market, further revision is required to be sure of the safety, efficacy, and quality of other nano theranostic for human use since the present standards might not be sufficient.

#### *Approach to Overcome the Challenges*

A lot of research efforts are required to overcome the biological barriers associated with lipidic cancer nanotheransotic. It is important to have a profound understanding of the correlation of disease heterogeneity and patient biology with nanomedicine, which is also the prime reason for the failures of promising nanoformulation in clinical trials. One of the strategies for mitigating clinical translation failures is the arduous assessment of nanoformulation in various animal models before starting the clinical trials. Expedient information regarding the suitability of lipidic theranostic nanomedicine can be obtained through preclinical studies before treatment and imaging of human subjects [127]. Nanotoxicology profiles consisting of standardized assay protocols for immunotoxicity, genotoxicity, and cytotoxicity should be to be implemented and followed to evaluate the potential risk in patients [128].

Academic laboratories are coming up with nanomedicine-based drug-delivery systems with great emphasis on new technological and scientific developments that succeed at a small scale. These laboratories normally know the technical issues, which occur in the industry for the commercializing processes. A strong collaboration among pharmaceutical companies and academic laboratory groups is required to be established to bridge this gap. There is a need to develop modified rules, which will be listed under good manufacturing practices that are suitable for large-scale synthesis of lipidic theranostic nanomedicines. To identify the key process and formulation variables for nanomedicine optimization and address batch-to-batch variation, optimization software such as Aspen (AspenTech,

Bedford, MA, USA) can be implemented in an industrial setting. For tightly controlled and robust manufacturing, this might be instrumental [129]. In conclusion, a positive outcome in manufacturing depends highly on how well the personnel is trained regarding the challenges, hurdles, and specificities related to the products. There is no doubt regarding the influence of lipidic theranostic nanomedicines on the health of humans in the clinic still. If the aforementioned lessons are applied in the early stages of development, it can help the producers prepare to develop efficient products.

#### **8. Conclusions**

The present manuscript brings forth the latest research updates in the field of lipidbased nanocarriers on cancer theranostic. The study findings are very motivating and strongly encourage a splendid exploration of lipidic nanocarrier in the area of cancer theranostic and make the outcomes clinically swappable. Moreover, this review provides a sound discussion over the impact of physicochemical attributes of lipid nanoparticles in improving in vivo performance of cancer theranostics. The review also discusses the limitations and suggests relevant solutions for the successful development of lipid-based cancer theranostic nanomedicines of improved attributes to pave their way to the clinics. Additionally, this review will provide a convenient guide for the researcher to know the significant findings of the recent studies carried out in the field of lipid nano carrier-based cancer theranostic in the last few years.

**Author Contributions:** Conceptualization and methodology, S.I.B., S.S.I.; software, J.A., M.Z.A.; formal analysis, S.A., J.A.; resources, M.Z.A., J.A.; writing—original draft preparation, S.S.I., S.I.B.; writing—review and editing, J.A., S.A., M.Z.A., P.R.V., visualization, J.A., M.Z.A., P.R.V.; project administration, S.S.I., J.A., W.A.M.; funding acquisition, W.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** Deanship of Scientific Research at King Saud University, Saudi Arabia: RG-1441-443.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group No. (RG-1441-443).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Chronicles of Nanoerythrosomes: An Erythrocyte-Based Biomimetic Smart Drug Delivery System as a Therapeutic and Diagnostic Tool in Cancer Therapy**

**Shamama Javed 1 , Sultan Alshehri 2,3 , Ambreen Shoaib 4 , Waquar Ahsan 5 , Muhammad Hadi Sultan 1 , Saad Saeed Alqahtani 4 , Mohsin Kazi 2 and Faiyaz Shakeel 2,\***

**Abstract:** Recently, drug delivery using natural biological carriers has emerged as one of the most widely investigated topics of research. Erythrocytes, or red blood cells, can act as potential carriers for a wide variety of drugs, including anticancer, antibacterial, antiviral, and anti-inflammatory, along with various proteins, peptides, enzymes, and other macromolecules. The red blood cell-based nanocarrier systems, also called nanoerythrosomes, are nanovesicles poised with extraordinary features such as long blood circulation times, the ability to escape immune system, the ability to release the drug gradually, the protection of drugs from various endogenous factors, targeted and specified delivery of drugs, as well as possessing both therapeutic and diagnostic applications in various fields of biomedical sciences. Their journey over the last two decades is escalating with fast pace, ranging from in vivo to preclinical and clinical studies by encapsulating a number of drugs into these carriers. Being biomimetic nanoparticles, they have enhanced the stability profile of drugs and their excellent site-specific targeting ability makes them potential carrier systems in the diagnosis and therapy of wide variety of tumors including gliomas, lung cancers, breast cancers, colon cancers, gastric cancers, and other solid tumors. This review focuses on the most recent advancements in the field of nanoerythrosomes, as an excellent and promising nanoplatform for the novel drug delivery of various drugs particularly antineoplastic drugs along with their potential as a promising diagnostic tool for the identification of different tumors.

**Keywords:** nanoerythrosome; nanoerythrocyte; nanovesicles; biomimetic; cancer therapy; diagnostics; imaging agents

#### **1. Introduction**

Erythrocytes (ER) or the red blood cells (RBC) are the most abundant blood cells responsible for the transport of gases from lungs to the tissues [1]. Over the past two decades, they have been exploited to develop various biomimetic cell-based carrier systems, including resealed erythrocytes (RE), genetically engineered RBCs, as well as RBC-membrane coated nanoparticles (NP) (RBC-m-NP) for various biomedical applications. Their transition from preclinical studies to clinical stage is a proof of being smart and effective enough to be used as novel drug delivery vehicles. Other than erythrocytes, other blood cells such as platelets and leukocytes can also be used as carrier system for a variety of drugs [2].

**Citation:** Javed, S.; Alshehri, S.; Shoaib, A.; Ahsan, W.; Sultan, M.H.; Alqahtani, S.S.; Kazi, M.; Shakeel, F. Chronicles of Nanoerythrosomes: An Erythrocyte-Based Biomimetic Smart Drug Delivery System as a Therapeutic and Diagnostic Tool in Cancer Therapy. *Pharmaceutics* **2021**, *13*, 368. https://doi.org/10.3390/ pharmaceutics13030368

Academic Editor: Hassan Bousbaa

Received: 21 February 2021 Accepted: 8 March 2021 Published: 10 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

All these cell-based drug carrier systems are superior and have various advantages over other drug-delivery systems such as micro- and nano-particles in terms of properties such as prolonged circulation time, biocompatibility, biodegradability, non-immunogenicity, ease of preparation, and flexibility in use [3], apart from improved patient compliance by extending the time of release of drugs from these systems. Escaping the rapid clearance from body and specificity for the target are other major advantages associated with such carriers [4]. These extraordinary features helped the carrier erythrocytes (C-ER) to emerge as potential drug-carrier systems for the therapy of cellular infections, cardiovascular disorders, cancer therapy, gene therapy, and other ailments [5]. Recently, several other cellbased drug delivery strategies such as bacterial ghosts, dendritic cells (DC) and genetically engineered stem cells have also emerged owing to the successful application of C-ERs [6].

Nanoerythrosomes (NER) are the C-ERs which are recognized as modern day, novel, and smart drug delivery systems associated with increased bioavailability, improved pharmacokinetics, and low toxicity [7]. As a physiological carrier, NERs can release the drug in circulation for weeks, have high loading capacity, are easy to be processed and have good ability to accommodate biologics, antigens, contrasting agents, peptides, proteins, enzymes, and macromolecules using different chemical and physical based methods [8,9]. Innumerable examples are available in the literature discussing the drug delivery by C-ERs for applications in drug targeting to the reticuloendotehlial system (RES), enzyme therapy and improvement in the delivery of oxygen to the tissues [10,11]. One of such example is the delivery of bovine serum albumin (BSA) as a model antigen, which can be loaded on the human erythrocytes and delivered to the RES [12]. C-ERs are the unique drug delivery system that is also known to be capable of modulating the immune responses [13]. Amongst different antineoplastic drugs, methotrexate, etoposide, doxorubicin, and carboplatin have been successfully encapsulated in these carriers previously [14]. C-ERs are shown to protect the carried therapeutics such as antibiotics, antineoplastics, corticosteroids, peptides, and enzymes from being inactivated by different endogenous factors. The three main mechanisms, by which these C-ERs work are prolongation of circulation half-life, slow drug release, and organ-specific targeting [15]. Regulatory aspects of carrier erythrocytes and industrial developments are evolving day by day. However, their production scalability, process validation and quality control is still challenging enough for successful industrialization [16].

This review focuses on the NERs which have emerged as a promising and versatile platform for the successful delivery of a number of drugs in particular the antineoplastic drugs. Recent advancements in the field of NERs along with the nanoformulations developed and used in the cancer and non-cancer therapy are covered. The diagnostic and imaging applications of NERs, as well as the recent patents and clinical trials associated with these carriers have also been discussed.

#### **2. Fabrication of NERs**

Various methods have been successfully utilized for the fabrication of NERs in order to enhance membrane permeability and cellular targeting of therapeutic agents. These methods are described below [17–20].

#### *2.1. Dilutional Hemolysis and Resealing Method*

Hemolysis and resealing method is commonly used method for drug loading into erythrocytes. In this technique, the erythrocytes are exposed to hypotonic solution (0.4% *w/v* sodium chloride) until it reaches a critical value of volume or pressure in order to rupture the cells. Then, resealing is carried out in order to restore osmotic properties [19]. Dilutional hemolysis utilizing hypotonic solution (0.4% *w/v* sodium hydroxide) at 0 ◦C and resealing at 37 ◦C has been utilized as cellular carriers for the successful delivery of low molecular weight drugs like β-glucosidase and β-galactosidase [20]. This technique is the simplest and fastest technique for the preparation of NERs. The main disadvantages of this

technique are low drug loading efficiency (1–8%) and considerable loss of hemoglobin and other cellular components [19,20].

#### *2.2. Preswell Dilutional Hemolysis Method*

The preswell dilutional hemolysis technique has been developed for the improvement of drug loading efficiencies. In this technique, an initial swelling of erythrocytes is achieved using slightly hypotonic solution, followed by low-speed centrifugation. The drug loading efficiencies of some drugs such as ibuprofen and thyroxin have been significantly increased using preswell dilutional hemolysis technique compared to dilutional hemolysis technique [17].

#### *2.3. Hypotonic Dialysis Method*

The main problems of dilution techniques is the low drug-loading efficiencies. This problem of such dilutional techniques can be overcome using hypotonic dialysis technique. In hypotonic dialysis technique, the lysis and resealing are carried out within dialysis tube. This technique offers high drug entrapment efficiency, high drug-loading efficiency, and high cells recovery compared to dilutional techniques. However, long processing time and special instruments are required for this technique. This method has been successfully utilized for the loading of various enzymes (asparginase, galactosidase, and glucoserebrosidase, etc.) and drugs (gentamicin, adriamycin, and furamycin, etc.) [17,21].

#### *2.4. Use of Red Cell Loader*

The instrument like red cell loader is used for the loading of non-diffusible drugs into erythrocytes. Using this technique, the drugs are loaded into small volume of blood within 2 h at room temperature under blood banking conditions. The drug loading up to 30% and cell recovery of 30–50% can be achieved using this technique [18].

#### *2.5. Isotonic Osmotic Lysis*

It is also known as osmotic pulse technique which can be achieved by chemical or physical means. The chemical which are used to achieve isotonic hemolysis are urea, polyethylene glycols, and ammonium chloride. This technique is not immune to the changes in the composition of membrane structures [17,18].

#### *2.6. Membrane Perturbation Technique*

This technique is based on the enhancement in the membrane permeability of erythrocytes when the cells are exposed under certain chemicals or drugs. The membrane permeability of erythrocytes was enhanced under exposure to an antibiotic amphotericin B. This technique has been applied to load an antitumor drug daunomycin into erythrocytes of rat and human. However, this technique is irreversible and hence not very popular [17,18].

#### *2.7. Lipid Fusion Technique*

This technique is used for the loading of drug-loaded lipid vesicles into erythrocytes. Drug-loaded lipid vesicles can be directly fused into human erythrocytes, which could result into exchange with a lipid-entrapped drug. This technique has been successfully applied for the loading of inositol monophosphate into human erythrocytes for the improvement of oxygen carrying capacity of the cells. However, the drug-loading capacity of this technique is too low (1%) [18,19].

Some other techniques such as extrusion, sonication, and electrical breakdown techniques have also been utilized for the loading of drugs into human erythrocytes [18].

#### **3. NERs as an Efficient Drug Delivery Tool**

The compendium of knowledge and several research studies available on the RBCbased carrier systems led to the development of more advanced RBC-membrane derived liposomes also called as NERs. It has opened a new vista of its futuristic application in

the field of nanomedicine with greater potentials. These are NPs often designated with other names such as functionalized-NPs, hybrid-NPs, biomimetic-NPs, and targeted-NPs in the literature [22–24]. The NERs, being nanosized derivatives of erythrocytes, exhibit important properties such as biodegradability, biocompatibility, excellent release profiles, and organ and site-specific targetability. The prolonged circulation time in blood for NERs is owing to the higher surface area-to-volume ratio than the parent erythrocytes (approximately 80 times higher). Extra care is necessary while handling parent erythrocytes for maximum drug loading. The hemoglobin and cytoplasmic contents of the erythrocytes are removed using a controlled hypertonic solution mediated hemolysis followed by the loading of therapeutic agents. This process of removal of cellular contents should be in such a controlled manner that the plasma membrane should not be disrupted and the cells are allowed to form seamless nanosized capsule. One erythrocyte can be fragmented into thousands of NERs depending upon the recovered cell percentage post removal of its organelles. The NERs are prepared from the hemoglobin (Hb)-free erythrocyte ghosts using sonication, extrusion, and electrical impulses techniques. Uniform nanosized NERs are obtained using extrusion technique which is the most efficient technique amongst all. The nanosizing technique, lysis media, and processing temperature have considerable impact on the development of stable and viable NERs-based formulations. Owing to the biomimetic properties of NERs, these can be utilized for the efficient encapsulation of peptides, enzymes, genetic materials, toxins, and contrasting agents which can be applied for the treatment of liver, lymph nodes, and spleen disorders, as well as various types of carcinomas [23–25]. Similar to liposomes, NERs are the nanovesicles prepared by the extrusion of RBC ghosts, are membrane-covered nanoscale containers, discrete spherical in shape, with unilamellar membrane of 4.5 µm thickness and having diameter up to 100 nm (Figure 1). However, the disadvantages associated with liposomes and monoclonal antibodies are not present in NERs and these show excellent colloidal stability in both buffer and serum at room temperature [25]. The compendium of knowledge and several research studies available on the RBCbased carrier systems led to the development of more advanced RBC-membrane derived liposomes also called as NERs. It has opened a new vista of its futuristic application in the field of nanomedicine with greater potentials. These are NPs often designated with other names such as functionalized-NPs, hybrid-NPs, biomimetic-NPs, and targeted-NPs in the literature [22–24]. The NERs, being nanosized derivatives of erythrocytes, exhibit important properties such as biodegradability, biocompatibility, excellent release profiles, and organ and site-specific targetability. The prolonged circulation time in blood for NERs is owing to the higher surface area-to-volume ratio than the parent erythrocytes (approximately 80 times higher). Extra care is necessary while handling parent erythrocytes for maximum drug loading. The hemoglobin and cytoplasmic contents of the erythrocytes are removed using a controlled hypertonic solution mediated hemolysis followed by the loading of therapeutic agents. This process of removal of cellular contents should be in such a controlled manner that the plasma membrane should not be disrupted and the cells are allowed to form seamless nanosized capsule. One erythrocyte can be fragmented into thousands of NERs depending upon the recovered cell percentage post removal of its organelles. The NERs are prepared from the hemoglobin (Hb)-free erythrocyte ghosts using sonication, extrusion, and electrical impulses techniques. Uniform nanosized NERs are obtained using extrusion technique which is the most efficient technique amongst all. The nanosizing technique, lysis media, and processing temperature have considerable impact on the development of stable and viable NERs-based formulations. Owing to the biomimetic properties of NERs, these can be utilized for the efficient encapsulation of peptides, enzymes, genetic materials, toxins, and contrasting agents which can be applied for the treatment of liver, lymph nodes, and spleen disorders, as well as various types of carcinomas [23–25]. Similar to liposomes, NERs are the nanovesicles prepared by the extrusion of RBC ghosts, are membrane-covered nanoscale containers, discrete spherical in shape, with unilamellar membrane of 4.5 µm thickness and having diameter up to 100 nm (Figure 1). However, the disadvantages associated with liposomes and monoclonal antibodies are not present in NERs and these show excellent colloidal stability in both buffer and serum at room temperature [25].

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 4 of 23

**3. NERs as an Efficient Drug Delivery Tool**

**Figure 1.** Preparation of nanoerythrocytes (NER) from normal erythrocytes (ER). **Figure 1.** Preparation of nanoerythrocytes (NER) from normal erythrocytes (ER).

#### **4. Applications of NERs in Cancer Therapy and Diagnosis**

#### *4.1. NERs in Cancer Therapy*

The important studies on NERs and their formulations for the application in cancer therapy are summarized in Table 1. NERs are artificial, synthetic, biomimetic, and presently the most investigated and sought after RBC cell-based carrier systems under this category. These RBC cell-based carrier systems have covered a path of progress from conven-

tional REs (microscale) to the most advanced NERs (nanoscale). These RBC-nanovesicles (RBC-NV) are fused onto the core NPs through extrusion, sonication, and electroporation techniques. Being biomimetic NPs, NERs attain specific functions including ligand recognition, tumor-targeting, longer blood circulation time, and ability to escape the immune system. These activities lead to the core-shell interactions, mimicking the source cells, and improving the therapeutic efficacy of drugs through specific delivery and enhanced drug-accumulation inside the tumor [26]. NERs marked the beginning of an unparalleled approach towards the therapy of a number of diseases including cancer owing to multiple advantages such as small size, ability to encapsulate antineoplastic drugs, and organ-specific targeting which further gave them added advantages over their other carrier counterparts in cancer nanomedicine. The surface of NERs can be modified using tumor-targeting ligands, which further improve the drug efficacy and optimize drug pharmacokinetics.

The loading and delivery of antineoplastic drugs using various pharmaceutical and nanotechnological engineering techniques is one of the most important applications of NERs. The unique techniques of surface modification and functionalization provide additional attractive feature to this delivery system and further increase its potential in cancer therapy (Figure 2). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 23

**Figure 2.** Role of NERs in cancer therapy. **Figure 2.** Role of NERs in cancer therapy.

The poor drug loading, low anti-tumor efficacy, and rapid clearance from the blood circulation are the major challenges faced by the conventional anti-cancer nanocarriers. The most challenging part is the high uptake of nanocarriers by the liver and spleen because of RES. The majority of nanocarriers injected intravascularly end up in the liver causing hepatotoxicity [29]. The NERs, as an innovative drug delivery system, provides with high drug loading capacity, specificity in targeting tumor cells, and synergistic anticancer sensitization using human erythrocyte membrane as delivery platform. This novel technology can be used in treating and preventing cancer metastasis such as breast cancer Numerous studies have been performed in the field of cancer therapy using NERs over the last two decades and multitude of its medical applications in other ailments diversify their applications in the arena of drug delivery as therapeutic, diagnostic, theragnostics, and imaging tool [27]. The nanomedicines have the ability to deliver the drugs directly to the individual organs to avoid the adverse or side effects; however, the drug delivery by conventional nanocarriers is limited by difficulty in delivering to most target organs and dire need of the affinity moieties [28].

metastasis, mainly by blocking the circulation of metastatic cancer cells by inhibiting angiogenesis and capturing circulating endothelial progenitors that are recruited to the tumor. They also physically block (infarction) the capillaries of the tumor or the metastasis. The inhibition of molecular pathways of the metastatic invasion and cancer growth is the latest therapeutic approach for cancer metastasis. The physical blocking of metastasis and angiogenesis by incorporating tumor-and angiogenesis-specific ligands such as antibodies, small molecules and peptides into the plasma membrane of erythrocytes is very novel [30]. The poor drug loading, low anti-tumor efficacy, and rapid clearance from the blood circulation are the major challenges faced by the conventional anti-cancer nanocarriers. The most challenging part is the high uptake of nanocarriers by the liver and spleen because of RES. The majority of nanocarriers injected intravascularly end up in the liver causing hepatotoxicity [29]. The NERs, as an innovative drug delivery system, provides with high drug loading capacity, specificity in targeting tumor cells, and synergistic anti-

Polymeric-NPs as intravascular drug delivery vehicles have been studied extensively

delivery of various anticancer pharmaceutical agents. These anticancer drugs, when administered intravenously, cannot reach the tumor site efficiently owing to their weal specificity which results into lower therapeutic potential and serious side effects. One of the major reasons of multidrug resistance (MDR) and thereby chemotherapy failure is the efflux of anticancer drugs mediated by P-glycoprotein (P-gp). This problem is overcome to an extent when these anticancer agents are delivered to the tumors in combination with P-gp inhibitors and this has proven to be an effective strategy [31]. An organ-specific or site-specific nano-drug delivery system based on human RBCs is a newer and effective

strategy of the present decade (Figure 3).

cancer sensitization using human erythrocyte membrane as delivery platform. This novel technology can be used in treating and preventing cancer metastasis such as breast cancer metastasis, mainly by blocking the circulation of metastatic cancer cells by inhibiting angiogenesis and capturing circulating endothelial progenitors that are recruited to the tumor. They also physically block (infarction) the capillaries of the tumor or the metastasis. The inhibition of molecular pathways of the metastatic invasion and cancer growth is the latest therapeutic approach for cancer metastasis. The physical blocking of metastasis and angiogenesis by incorporating tumor-and angiogenesis-specific ligands such as antibodies, small molecules and peptides into the plasma membrane of erythrocytes is very novel [30].

Polymeric-NPs as intravascular drug delivery vehicles have been studied extensively in past but their applications are beset by rapid clearance from the circulation by RES. This led to the development of NERs as novel cancer drug delivery systems for the optimal delivery of various anticancer pharmaceutical agents. These anticancer drugs, when administered intravenously, cannot reach the tumor site efficiently owing to their weal specificity which results into lower therapeutic potential and serious side effects. One of the major reasons of multidrug resistance (MDR) and thereby chemotherapy failure is the efflux of anticancer drugs mediated by P-glycoprotein (P-gp). This problem is overcome to an extent when these anticancer agents are delivered to the tumors in combination with P-gp inhibitors and this has proven to be an effective strategy [31]. An organ-specific or sitespecific nano-drug delivery system based on human RBCs is a newer and effective strategy of the present decade (Figure 3). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 7 of 23

**Figure 3.** Summary of red blood cell (RBC)-based anticancer nanoformulations (anti-cancer-loaded-NERs) along with their mechanisms. **Figure 3.** Summary of red blood cell (RBC)-based anticancer nanoformulations (anti-cancer-loaded-NERs) along with their mechanisms.

The anticancer drug delivery using NERs was first carried out by a team of researchers two decades ago in 1994, when they explored the anticancer drug daunorubicin (DAU) and prepared the DAU-NERs for enhanced antineoplastic potential. DAU was linked covalently to the NERs using glutaraldehyde as homobifunctional linking arm, and it was observed that the cytotoxicity of conjugated drug was higher as compared to the free drug when assessed on P388D1 cancer cell line [32]. Results indicated that these DAU-NER conjugates could potentially be utilized as drug carriers in CDF1 leukemia tumors. The mechanism behind the cellular uptake was neither the diffusion through cell membrane The anticancer drug delivery using NERs was first carried out by a team of researchers two decades ago in 1994, when they explored the anticancer drug daunorubicin (DAU) and prepared the DAU-NERs for enhanced antineoplastic potential. DAU was linked covalently to the NERs using glutaraldehyde as homobifunctional linking arm, and it was observed that the cytotoxicity of conjugated drug was higher as compared to the free drug when assessed on P388D1 cancer cell line [32]. Results indicated that these

> nor endocytosis but free DAU was found to slowly release by hydrolysis of glutaraldehyde linking arm, producing a high concentration of free DAU in the cells vicinity for a

> The biodistribution of 125I-NERs purified by dialysis followed by intravenous (IV) and intraperitonial (IP) injections in CD1 mice was investigated in another study. NERs were rapidly (less than 30 min) removed from the systemic circulation after IV administration, however, the IP administration showed a higher uptake by liver and spleen, accumulation in lungs and marked activity was observed in the inguinal lymph nodes after 2 h of the

> Earlier, the surface modification of NPs could be achieved using polymers such as poloxamines, poloxamers and polyethylene glycols, to avoid opsonization, but now, these NPs could be anchored to the surface of RBCs to escape the clearance by RES as the RBCs are able to do so. This leads to a novel approach in the intravascular drug delivery system as RBC-anchored NPs. The RBC-anchored polymeric NPs were prepared using polystyrene NPs modified with carboxyl groups (110, 220, 450, and 1100 nm in diameter). An

administration [35].

DAU-NER conjugates could potentially be utilized as drug carriers in CDF1 leukemia tumors. The mechanism behind the cellular uptake was neither the diffusion through cell membrane nor endocytosis but free DAU was found to slowly release by hydrolysis of glutaraldehyde linking arm, producing a high concentration of free DAU in the cells vicinity for a long period of time [33], and phagocytosis of DAU-NER complex was not involved [34]. The biodistribution of 125I-NERs purified by dialysis followed by intravenous (IV) and intraperitonial (IP) injections in CD1 mice was investigated in another study. NERs were rapidly (less than 30 min) removed from the systemic circulation after IV administration, however, the IP administration showed a higher uptake by liver and spleen, accumulation in lungs and marked activity was observed in the inguinal lymph nodes after 2 h of the administration [35].

Earlier, the surface modification of NPs could be achieved using polymers such as poloxamines, poloxamers and polyethylene glycols, to avoid opsonization, but now, these NPs could be anchored to the surface of RBCs to escape the clearance by RES as the RBCs are able to do so. This leads to a novel approach in the intravascular drug delivery system as RBC-anchored NPs. The RBC-anchored polymeric NPs were prepared using polystyrene NPs modified with carboxyl groups (110, 220, 450, and 1100 nm in diameter). An improved in vivo circulation lifetime was achieved using RBC-anchored NPs [36]. In another study by the same research group, RBC-anchored NPs were eventually get detached from RBCs owing to the shear forces and subsequently get cleared up in the liver and spleen [37]. The surface modifications of NERs is possible with glutaraldehyde, antibodies, and carbohydrates (sialic acid), which improve their target specificity and prolong their in vivo circulation time, helps escape the RES and protects the drug from the inactivation by endogenous chemicals and enzymes [38,39]. Based on this approach, a top-down biomimetic approach was reported utilizing particle functionalization. The fluorophore-loaded biomimetic NPs were prepared using poly (*d,l*-lactide-co-glycolide) (PLGA) polymer and fused with RBCderived vesicles. The prepared RBC-fused biomimetic NPs were injected in mice. Results revealed superior circulation half-life in cases of biomimetic-NPs as compared to control group-NPs which were coated with conventional synthetic stealth materials. An increased particle retention time of 72 h in the blood was observed which proved the approach as very unique and effective in the NPs functionalization [40]. In another study, RBC-membranecloaked polymeric NPs were prepared by lipid-insertion technique utilizing fluorescein isothiocyante (FITC)-polyethylene glycol (PEG)-lipid. The FITC-PEG-lipid NPs were functionalized with folate and nucleolin-targeting aptamer AS1411 showed a receptor-specific targeted delivery when tested against the cancer cell lines models [41]. Another promising integration of cell-based drug delivery and nanotechnology was seen when RBCs were linked to iron oxide-NPs pre-coated with chlorine-e6 and a well-known chemotherapeutic drug, doxorubicin (DOX) was loaded for an imaging-guided combined chemotherapyphotodynamic therapy of carcinomas. Interestingly, the marked synergism and excellent therapeutic results were observed in animal tumor model [42]. In another report, the oxygen self-enriched biomimetic RBCs were prepared by cloaking the albumin-NPs containing indocyanine green (ICG) and perfluorocarbon (PFC) with RBC membranes. The rapid clearance of conventional nanocarriers by RES and low singlet oxygen quantum (0.08) of ICG often resulted into limited phytotherpaeutic efficacy. The prepared oxygen self-enriched RBCs had long circulation time, high oxygen carrying capacity, and biomimetic properties ideal for clinical cancer phototherapy treatment [43]. Another novel NERs-ghost delivery system was developed to transport sodium transhinone-IIA sulphonate (STS-Nano-RBC) for IV use in rats. These nanosystems had narrow particle size distribution, good stability for 21 days, and better loading efficiency as compared to conventional STS injections in vivo [44].

BackgroundIt is now evident that the adverse effects associated with the conventional cancer therapy (chemotherapy, surgery, and radiotherapy) can effectively be addressed by applying the nanomedicine approach. Nano-engineering makes targets achievable and with diverse surface functionalization methods, nowadays RBCs are not only targeting

drug carriers but therapeutic and imaging diagnostic tools as well, what we commonly term as theranostics [45]. Recently, RBC membrane-loaded with hydrophobic drug camptothecin were prepared which were non-covalently labeled with amphiphilic fluorophore nanovesicles (NVs). These NVs were overall stable, nonphagocytic and exhibited minor stimulation of macrophages for cytokine release. Intravenously injected NVs in balb/c mice exhibited better retention over 48 h and minimal accumulation in vital organs such as heart, liver, and kidneys [46]. NE-membrane-chaperoned 5-fluorouracil (5-FU) liposomes were also prepared as biomimetic delivery system to target hepatocellular carcinoma cell lines. Liposomes of 5-FU were prepared by thin film hydration method using dipalmitoyl phosphatidylcholine (DPPC) and cholesterol. NE-membrane-chaperoned 5-FU liposomes were prepared by extrusion technique. The drug-targeting capability of NE-membranechaperoned liposomes was higher than conventional 5-FU solution [47]. The most recent advances in this field include antigen delivery systems using NERs in which the tumor antigens are loaded onto the NERs using fusion tumor cell membranes. This nanosystem was shown to markedly inhibit the tumor growth in B16F10 and 4T1 tumor models in vivo [48]. A new NER-tailoring technique evolved recently, wherein, the NERs in the size range of 200 to 300 nm were produced by addition of phospholipids DPPC to ghost RBCs [49]. In yet other significant research, bio-hybrid microswimmers were developed which had the integration of motile-microorganisms with artificial cargo carriers to revolutionize targetedcargo delivery. In this study, genetically-engineered flagellated *Escherichia coli* species were integrated with RBC-derived NERs through conjugation of streptavidin-modified microorganisms with biotin-modified NERs using non-covalent streptavidin-biotin interactions. The high fabrication efficiency and good motility performance was achieved with these personalized biohybrid microswimmers, as shown in Figure 4 [50]. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 9 of 23 [49]. In yet other significant research, bio-hybrid microswimmers were developed which had the integration of motile-microorganisms with artificial cargo carriers to revolutionize targeted-cargo delivery. In this study, genetically-engineered flagellated *Escherichia coli* species were integrated with RBC-derived NERs through conjugation of streptavidinmodified microorganisms with biotin-modified NERs using non-covalent streptavidinbiotin interactions. The high fabrication efficiency and good motility performance was achieved with these personalized biohybrid microswimmers, as shown in Figure 4 [50].

**Figure 4.** Biohybrid microswimmers using genetically modified *E. coli* bacterium and NERs for targeted cargo-delivery (Image reproduced from reference [50] which was published under a creative commons attribution (CC BY) license*. 4.2. NERs in Immunotherapy*  **Figure 4.** Biohybrid microswimmers using genetically modified *E. coli* bacterium and NERs for targeted cargo-delivery (Image adopted from reference [50], APL Bioeng, 2020, which was published under a creative commons attribution (CC BY) license).

#### The main focus of cancer immunotherapy is to manipulate the patient's own immune system to recognize and destroy the cancer cells. The NPs-based drug delivery systems *4.2. NERs in Immunotherapy*