Earlier, we have developed citrate-based biomaterials, known as biodegradable photoluminescent polymers (BPLPs) that possessed a strong and tunable photoluminescence phenomenon; and demonstrated their potential use in bioimaging, drug delivery and tissue engineering (Yang et al., 2009). Unlike other imaging agents that are not degradable, BPLPs are created from biocompatible monomers via a convenient thermal polycondensation reaction and shown to have controlled degradability properties. However, the main challenge of using BPLPs for nanoparticle fabrication was associated with their low molecular weight, which resulted in nanoparticle aggregation in physiological conditions, hence limiting their use as an imaging probe. To overcome this, we synthesized new polymers by incorporating BPLPs into the widely used biodegradable polylactones, referred to as biodegradable photoluminescent polylactones (BPLPLs) that showed higher molecular weight, improved mechanical strength, and favorable processability over BPLPs (Xie et al., 2014; Hu et al., 2016). The intrinsic and stable fluorescent property of BPLPs is well-preserved in BPLPLs. Furthermore, the BPLPLs fluorescence emission ranging from blue to red can be adjusted by varying different amino acids in the syntheses of BPLPs (Yang et al., 2009; Xie et al., 2017).

In this research, we developed three different nanoparticles based on BPLPLs including BPLP-co-poly (L-lactic acid) (BPLPL-PLLA) and BPLP-co-poly (lactic-co-glycolic acid) copolymers with lactic acid and glycolic acid ratios of 75:25 (BPLPL-PLGA75:25) as well as 50:50 (BPLPL-PLGA50:50). Furthermore, we have characterized for their physical properties and biocompatibility with the blood cells and endothelial cells and investigated for their bioimaging applications. Our preliminary characterization studies would help us to identify a suitable BPLPL-based material to synthesize theranostic NPs that can be utilized both as an imaging agent to track the EC delivery and as a vascular drug carrier to promote in situ reendothelialization post arterial injury.

#### EXPERIMENTAL PROCEDURES

#### Materials

Synthesis of BPLPLs such as BPLPL-PLLA (1:100), BPLPL-PLGA50:50 (1:100), BPLPL-PLGA75:25 (1:100) was described previously (Xie et al., 2014; Hu et al., 2016). The ratio of 1:100 represents the feeding molar ratio of BPLP either with lactic acid or a combination of lactic acid and glycolic acid. PLGA50:50 of molecular weight 55–65 kDa was purchased from Akina, Inc. (West Lafayette, IN). Other reagents including bovine serum albumin (BSA) and polyvinyl alcohol (PVA) of molecular weight 31–50 kDa were brought from Sigma-Aldrich (St. Louis, MO). MTS reagent (CellTiter 96 R AQueous One Solution Cell Proliferation Assay) and Pierce BCA protein assay were obtained from Promega (Madison, WI) and ThermoFisher Scientific (Grand Island, NY), respectively. OxiSelectTM Intracellular Nitric Oxide (NO) Fluorometric Assay Kits were purchased from CellBioLabs, Inc. (San Diego, CA). Furthermore, human umbilical vein endothelial cells (HUVECs) was purchased from American Type Culture Collection (ATCC, Manassas, VA), while the culture media (Vasculife Basal Medium) and supplemental kits (Vasculife VEGF Lifefactors) were purchased from Lifeline Cell Technology (Frederick, MD). Other chemicals, if not specified were purchased from Sigma Aldrich.

#### Synthesis of BPLPL-Based Nanoparticles

BSA was selected as the model protein to be encapsulated into BPLPL nanoparticles, which were synthesized by a standard double emulsion technique. For this procedure, BSA solution (20 mg of BSA dissolved in 0.2 ml of DI water) was emulsified into 2% (w/v) BPLP-polylactones solution prepared in 5 mL of chloroform and sonicated. This primary emulsion was added drop wise into 12 mL of 5% (w/v) PVA and sonicated again at 30 W for 5 min. Following the overnight stirring to evaporate organic solvents, the nanoparticles were washed and isolated by centrifugation at 15,000 rpm for 30 min, and protein (BSA) loaded BPLPL based NPs were collected via freeze-drying. Blank BPLPL NPs as well as PLGA50:50 NPs were also fabricated using similar procedures without adding BSA to be utilized for in vitro cells- and blood-based studies.

#### Physical Characterization of BPLPL-Based Nanoparticles

The nanoparticles were characterized for their particle size, polydispersity and zeta potential via a dynamic light scattering (DLS) method using Zeta PALS zeta potential analyzer (Brookhaven Instruments, Holtsville, NY). The size and morphology of the nanoparticles were also observed using transmission electron microscopy (TEM). The stability of particles was determined by observing the variation in their size while suspended in various formulations such as DI water, saline (0.9% sodium chloride solution), 10% Fetal Bovine Serum (FBS, Atlanta Biological, Lawrenceville, GA), or simulated body fluid with similar composition of blood plasma, prepared as described previously Marques et al., 2011). The particles were incubated at 37◦C and their sizes were measured using DLS every 12 h up to 3 days. Furthermore, the amount of BSA encapsulated into BPLPL-based particles was estimated based on unentrapped BSA in PVA solution after centrifugation. The percentage of loading efficiency was calculated as actual amount of BSA loaded with respect to the initial amount of BSA used to prepare NPs. For the in vitro release study of BSA, 1 mg/ml of particle solution in PBS suspended in a 100 kDa dialysis bag (Spectrum Laboratories Inc., Rancho Dominguez, CA) was dialyzed against phosphate buffer saline (PBS) solution. At each predetermined time points, 1 ml of dialysate solution was collected and replaced with fresh PBS solution. BSA content in the collected solution was quantified using BCA protein assays following manufacturer's instructions, and cumulative BSA release over the time was analyzed based on BSA standards. In vitro degradation of nanoparticles in DI water was analyzed over a period of 4 weeks. Briefly, NPs was suspended in DI water and incubated at 37◦C for predetermined times. At each time point, particles were collected and freeze dried. The degradation was determined based on the remaining mass of NPs.

#### In vitro Cell Studies of BPLPL-Based Nanoparticles Cytocompatibility

To evaluate the cytotoxicity of BPLPL-based nanoparticles with human umbilical vein endothelial cells (HUVEC), cells were seeded in 96 well plates at a seeding density of 30,000 cells/cm2 and incubated in 37◦C for 24 h. Following incubation, cell culture media was replaced with increasing concentrations of nanoparticle suspension (in media) for 24 h. The cells were then washed and incubated with MTS assay reagents for 3 h. Absorbance readings was measured at 490 nm using UV-Vis spectrophotometer (Infinite M200 plate reader, Tecan, Durham, NC), and the percent of cell viability was determined with respect to untreated cells.

#### Cellular Uptake

The efficiency of HUVECs to internalize BPLPL-based nanoparticles was determined. Briefly, HUVECs of density 30,000 cells/cm2 were initially seeded in to 96 well plates and allowed to attach for 24 h. The cell culture media was then replaced with nanoparticle suspensions of various concentrations, and the plates were incubated for 4 h. After treatment, cells were washed with PBS and lysed with 1% Triton X-100 for 30 min at 37◦C, and lysate was utilized to measure the nanoparticles' fluorescence intensities at excitation and emission wavelength of 377 and 431 nm, respectively. These measurements were analyzed against a nanoparticle standard. These fluorescence intensity values were then normalized with the total protein content per sample using BCA assays following manufacturer's instructions. In parallel, the nanoparticle interactions with endothelial cells were imaged using a fluorescence microscope under FITC channel.

#### Cellular Functionality

HUVEC functionality in the presence of BPLPL-based nanoparticles was determined based on nitric oxide (NO) production. Nanoparticles (1 mg/ml) were incubated with cells for 24 h, following which nitric oxide production of exposed cells was quantified using Intracellular Nitric Oxide Fluorometric Assay kits (Cell Biolabs, Inc., San Diego, CA) following the manufacturer's instructions. In brief, a NO fluorometric probe (provided with the kit) enters the cells and deacetylates by intracellular esterase to a non-fluorescent intermediate, which is rapidly oxidized by nitric oxide into a fluorescent triazolofluorescein analog. The fluorescence intensity is proportional to NO levels within the cell cytosol, which can be quantified at Kuriakose et al. BPLPL-Based Theranostic NPs

a wavelength of 480 nm (excitation)/530 nm (emission) using UV/vis spectrophotometer (Infinite M200 plate reader, Tecan, Durham, NC). Cells grown on tissue culture plates without any treatment served as control.

# In vitro Blood Studies of BPLPL-Based Nanoparticles

#### Blood Collection

Whole blood was drawn from healthy adult volunteers into acid citrate dextrose anticoagulant tubes (ACD, Solution A; BD Franklin Lakes, NJ). Consent from the volunteers was obtained prior to the blood collection, and all the procedures strictly adhered to the IRB standards approved at the University of Texas at Arlington.

#### Whole Blood Clotting Kinetics and Hemolysis

Briefly, in hemolysis, 10 µL of various concentrations of nanoparticles ranging from 0 to 1,000µg/ml were incubated with 200 µl of blood for 2 h at 37◦C. The nanoparticles were centrifuged at 1,000 rpm for 5 min and absorbance of the supernatant was obtained using UV-Vis Spectrophotometer at a wavelength of 545 nm. Blood diluted in DI water served as the positive control, whereas saline diluted blood as the negative control for hemolysis studies. Percentage of hemolysis due to each sample was quantified based on Equation (1). In the whole blood clotting study, we studied the effects of particles on normal blood clotting kinetics, which was measured as blood clotting index (BCI). Here, whole blood initially activated by adding 0.01 M of calcium chloride, and 50 µL of activated blood was then treated with 10 µL of 0.5 mg/ml of nanoparticles for predetermined time points. At each time point, 1.5 ml of DI water was added to lyse the un-clotted blood, and absorbance of supernatant was measured at 540 nm. Untreated blood served as a control. The absorbance of whole blood (without any addition of calcium chloride) in water at 540 nm was applied as a reference value. The BCI can be quantified from Equation (2). TABLE 1 | Size, charge, polydispersity of BPLPL based NPs.

with 2.5% glutaraldehyde (Electron Microscopy Science, 16536- 15) for overnight, post fixed with 1% Osmium tetroxide in 0.1 M Cacodylate buffer (Electron Microscopy Sciences, 19150) for 1 h, dehydrated with graded series of ethanol (50, 75, 95, 100%) for 15 min at each step, and further dried using varying ratios of hexamethyldisilane (HMDS) in ethanol (1:2, 1:1, 2:1) for 15 min at each step. Finally, the films were dried using 100% HMDS for 30 min and then sputter-coated with the silver for SEM. In addition, after incubating PRP with polymer films for 1 h, 5 µL of suspension was collected and incubated with saturating concentrations of CD42b-PE (platelet marker) and PAC1- FITC (activated glycoprotein GP IIb/IIIa receptor marker) for 20 min. The antibodies were obtained from BD Biosciences. The platelets were fixed with 1% paraformaldehyde for 2 h in 4◦C and were analyzed on BD LSRII flow cytometer. At least 10,000 events per sample were analyzed and identified based on their forward and side scattering characteristics and by positive staining with anti-CD42b-PE antibodies. The percentage of GpIIb/IIIa expressing platelets was calculated relative to the total number of platelets (CD42b positive cells).

#### Statistical Analysis

All the experiments were performed with n = 3–6 if not specified. Data were expressed as mean ± SEM. The statistical analysis was assessed using ANOVA followed by post-hoc Pairwise Multiple Comparisons using Holm–Sidak method on GraphPad Prism

$$\text{Hemolyis} \left( \% \right) = \frac{\text{Absorbance of sample} - \text{Absorbance of negative control}}{\text{Absorbance of positive control} - \text{Absorbance of negative control}} \tag{1}$$

$$\text{BCI} = \frac{\text{Absorbance of blood in contact with samples at set time points at } 545 nm}{\text{All } \text{In} \quad \text{In} \quad \text{In} \quad \text{In} \quad \text{In} \quad \text{In} \quad \text{In} \quad \text{In} \quad \text{In}} \tag{2}$$

Absorbance of whole blood in water at 545nm at time 0

#### Platelet Adhesion and Aggregation

We further investigated the hemocompatibility of BPLPL materials based on the platelet adhesion and activation. For this study, platelet rich plasma (PRP) was collected by centrifuging whole blood at 190 g for 12 min. PRP was incubated with BPLPL films for 1 h at 37◦C under static conditions. After 1 h, films were rinsed carefully with PBS and attached platelets were lysed using cell lysis solution for 1 h. The LDH release corresponding to platelet adhesion was quantified by detecting the amount of lactate dehydrogenase (LDH) present in the lysate solution using CytoTox 96 R Non-Radioactive Cytotoxicity Assays according to the manufacturer's instructions. Glass served as the positive control for comparison. The morphology of platelet adhesion on the polymer films were also visualized using scanning electron microscopy (SEM) imaging. Briefly, platelets on films were fixed (GraphPad Software Inc., CA). A significant difference was considered where P-values appeared <0.05.

#### RESULTS

#### Physical Characterization of BPLPL-Based NPs

DLS results show that the BPLPL-PLGA50:50, BPLPL-PLGA75:25 and BPLPL-PLLA based nanoparticles suspended in DI water have hydrodynamic sizes of 157, 149, and 145 nm, respectively (**Table 1**).

The polydispersity values ranging from 0.10 to 0.17 suggest the particles were well-dispersed. TEM images of BPLPL nanoparticle also confirms the uniform distribution of NPs with smooth and spherical morphology (**Figures 1A–C**). Zeta potential for BPLPL-PLGA 50:50, BPLPL-PLGA 75:25 and BPLPL-PLLA based nanoparticles were −24.7, −23.9, and −21.9 mV, respectively, suggesting that the particles might be stable in physiological solutions (Honary and Zahir, 2013). In addition, the stability of BPLPL based nanoparticles was evaluated at various formulations including DI water, 10% FBS, 0.9% saline and simulated body fluid by recording the nanoparticles diameter at fixed time intervals. We have observed that BPLPL-PLGA NPs were stable in all formulations for 48 h with no significant aggregation or change in size. Although BPLPL-PLLA NPs remained relatively stable in DI water, saline and serum, they tended to have some aggregation in simulated body fluid at 48 h (**Figure 2**).

To determine if these nanoparticles could be utilized for drug delivery applications, their drug release kinetics and degradation studies were conducted. BSA was chosen as the model growth factor. BPLPL-PLGA 50:50, BPLPL-PLGA 75:25 and BPLPL-PLLA showed a loading efficiency of 70, 69, and 77%, respectively. **Figure 3A** showed that both BPLPL-PLGA nanoparticles could release ∼50% content within 24 h, and the complete release was achieved in 7 days. On the other hand, BPLPL-PLLA nanoparticles demonstrated comparatively lower release kinetics, and only 50% BSA release was achieved in 2 weeks. The in vitro degradation study also emphasizes the role of polymer composition on particle behavior (**Figure 3B**), where BPLPL-PLGA particles showed a similar degradation rate and almost ∼80% degraded in 4 weeks. However, BPLPL-PLLA degraded slowly with only ∼30% lost in weight when observed after 4 weeks. Furthermore, all BPLP-cys-polylactone based nanoparticles showed maximum excitation and emission wavelength at 374 and 441 nm, respectively (**Figure 3C)**.

## In vitro Cell Studies With BPLPL-Based Nanoparticles

Cytocompatibility evaluation of BPLPL-based NPs at various concentrations after 24 h incubation with HUVECs was conducted. Accordingly, BPLPL NPs at all concentrations ranging from 50 to 1,000µg/ml was shown to be compatible with HUVECs with >80% viable cells after NP exposure (**Figure 4A**). In addition, the functional status of endothelial cells in the presence of BPLPL-based NPs was studied by assessing the nitric oxide (NO) production. Accordingly, we noted that NO production by HUVECs treated with BPLPL-PLLA NPs at 1,000µg/ml for 24 h was significantly lower than that of cells treated with BPLPL-PLGA NPs and untreated cells (**Figure 4B**). On the other hand, NOS activity quantified for HUVECs in the presence of both BPLPL-PLGA50:50 and BPLPL-PLGA75:25 demonstrated no negative effects on cellular function.

Lastly, the uptake of BPLPL-based NPs by vascular endothelial cells (HUVECs) was studied by incubating cells with various concentrations of NPs over 4 h. All NP formulations showed dose-dependent cellular uptake up to a concentration of 1,000µg/ml (**Figure 5A**). Fluorescence images also showed the internalization of BPLPL-based nanoparticles by endothelial cells and their subsequent localization in cytoplasmic region of cells after 4-h incubation with particles (**Figure 5B**).

# Hemocompatibility of BPLPL-Based Nanoparticles

Hemocompatibility of nanoparticles was determined based on whole blood clotting kinetics, hemolysis study, and platelet responses. Blood clotting time reflects the thromboresistance property of nanoparticles, and high thromboresistance (Blood clot index, BCI value) means high blood compatibility. All BPLPL-based nanoparticles showed similar BCI values with each other and when compared to untreated blood samples (**Figure 6A**). This suggests that particles have no significant effect on the normal blood clotting kinetics. Furthermore, BPLPLbased NPs at all tested concentrations proved to be nonhemolytic with a maximum of 0.4% which was well within the standardized ISO values for non-hemolytic materials, which is 0–2% (Cerda-Cristerna et al., 2011) (**Figure 6B**). To assess the compatibility of BPLPL-based materials with platelets, BPLPL films were incubated with PRP at 37◦C for 1 h. Based on the analysis, BPLPL-PLLA showed a significantly higher number of platelets adhered onto its surface than those of BPLPL-PLGA counterparts (**Figure 7A**). As platelets get activated, Pselectin translocate from intracellular granules to the external membrane, whereas fibrinogen aggregates platelets by bridging glycoprotein GPIIb/IIIa between adjacent platelets (Merten and Thiagarajan, 2000). Based on flow cytometric analysis, significant amount of platelet activation was seen on the glass surface as a positive control (**Figure 7B**). The amount of activation seen on BPLPL-PLGA surface was similar to those on PLGA surfaces. In concordance to these observations, SEM images also showed significantly higher platelet attachment on glass and BPLPL-PLLA surfaces than others. Closer observation of these images (**Figure 7C**) shows that platelets are spreading and aggregating on glass and BPLPL-PLLA surfaces. Other surfaces such as PLGA 50:50, BPLPL-PLGA 75:25, and BPLPL-PLGA 50:50 also presented a platelet shape change representing the early stage of platelet activation, which was characterized by transformation from discoid to spheroid form with small bulbous protrusions distributed over the platelet surface (Zilla et al., 1987).

# DISCUSSION

Bioimaging holds huge potential in the field of drug delivery, tissue engineering and regenerative medicine. During the last decade, several functional polymers with imaging capabilities were investigated to understand the biological processes and their potential applications for image-guided surgery and therapy (Braeken et al., 2017; Zhang and Yao, 2018). The BPLP is one such novel biomaterial with intrinsic and excellent photoluminescent properties that can be used as a label-free in vivo imaging tool for disease detection and treatment. The components that are utilized to develop BPLPs including citric acid, amino acids, and aliphatic diols, which are all commonly used in many FDA-regulated devices (Yang et al., 2009; Li et al., 2017). Recently, a family of BPLPLs based on BPLP have been reported (Xie et al., 2014). This new class of materials can be utilized to fabricate

FIGURE 1 | Morphological characterization of BPLPL-based NPs. TEM images of (A) BPLPL-PLGA50:50 (1:100), (B) BPLPL-PLGA75:25(1:100), (C) BPLPL-PLLA (1:100) shows uniform sized and spherical morphology of nanoparticles. The scale bar represents 200 nm.

theranostic nanoparticles which can be tracked with a variety of microscopy techniques, including fluorescent microscopy, confocal laser scanning microscopy and two-photon microscopy (Li et al., 2017).

In our present work, we screened three different BPLPLbased nanoparticles (BPLPL-NPs) including BPLPL-PLLA, BPLPL-PLGA50:50, and BPLPL-PLGA75:25 to determine the most promising formulation that can be utilized for theranostic applications in treating cardiovascular diseases. Our characterization showed that most BPLPL-NPs maintained uniform, spherical morphology with 150 nm in diameter and higher negative zeta potential values compared to PLGA 50:50 nanoparticles due to inclusion of citric acid component in BPLP. This increased surface charged groups on BPLPLbased NPs not only provide additional functional motifs required for the conjugation of targeting ligands but also improve NPs stability in physiological fluids. To evaluate this, the diameter of BPLPL-NPs in various formulations including DI water, serum, saline, and simulated body fluid was monitored for 48 h. Accordingly, we found that most of BPLPL-NPs, except BPLPL-PLLA at 48 h in simulated body fluids were relatively stable with no signs of aggregations. It is plausible that with long-term incubation of BPLPL-PLLA in simulated body fluids alters the colloidal stability of particles due to enhanced interactions of these NPs with various salts and enzymes that constitute the solvent. Lazzari

et al. (2012) have observed similar aggregation behavior for PLLA NPs in simulated body fluids after prolonged incubation. In such instances, simple modifications of BPLPL-PLLA NPs surface with PEG chains might be able to prevent the docking of enzymes or ions on nanoparticle surfaces, thereby improving their colloidal stability in physiological conditions (Singh et al., 2017).

The presence of PLLA in BPLPs also seem to affect the protein loading efficiency, protein release kinetics and degradation profile of BPLPL-PLLA NPs. It was noted that BPLPL-PLGA

FIGURE 5 | Cellular uptake of BPLPL-based nanoparticles. (A) Nanoparticle uptake by endothelial cells (HUVECs) at various concentrations after incubation for 4 hr in 37◦C was quantified in terms of amount of nanoparticles relative to protein amount per sample. \$ and # represents significance with respect to BPLPL-PLGA50:50 NPs at concentrations of 100 and 250µg/ml, respectively; whereas \* represents significance between BPLPL-PLGA50:50 NPs and BPLPL-PLLA NPs. (B) Fluorescent images of nanoparticles internalized HUVECs at 60X, green represents nanoparticles and blue for cell nuceli. The scale bar is 25 µm in length. Graph plotted in terms of average ± SEM.

FIGURE 6 | Hemocompatibility of BPLPL-based NPs. NP compatibility in whole blood was assessed based on (A) clotting kinetic profile in presence of 1 mg/ml of NPs and (B) hemolysis after incubating with nanoparticles of various concentrations for predetermined timepoints. Graph plot as average ± SEM.

NPs released ∼50% of BSA within 24 h, whereas BPLPL-PLLA achieved a similar amount of BSA release within 2 weeks. In addition, BPLPL-PLLA NPs demonstrated a slower degradation rate with ∼30% lost weight compared to BPLPL-PLGA NPs which showed ∼80% degradation within 4 weeks. Hu et al. (2016) explained that BPLP incorporation into polylactones

could enhance water permeability, which in turn, accelerate the drug release rate and degradation of copolymers. Also, the percentage of hydrophilic glycolic acid in BPLPL would also attribute to faster degradation and thereby drug release from the particles. We speculate that the hydrophobic nature of PLLA, may have improved their protein encapsulation compared to its PLGA counterparts by forming a hydrophobic wall to retard BSA leakage into the outer water phase during the NP synthesis (Liu et al., 2006). Furthermore, the photoluminescent property of BPLP was retained in BPLPL-based NPs, with maximum excitation and emission wavelength at 344 and 441 nm, respectively. Previously, we have shown that depending on the amino acids used in BPLP syntheses, the fluorescence emission could been broadened up to 725 nm, highlighting the versatility of these polymers for biomedical imaging (Tran et al., 2009).

Following the physical and chemical characterization of BPLPL NPs, their cytotoxicity with endothelial cells was investigated. BPLPL NPs exhibited excellent cytocompatibility with >80% of HUVECs viability post treatment with NPs at all concentrations. Xie et al. (2014) also reported similar values for cell viability using 3T3 fibroblasts exposed to BPLPL-PLLA nanoparticles at concentrations ranging from 1 to 500µg/ml. Due to the presence of high number of carboxylic groups on the BPLP backbone, it was noted earlier about the reduced cell survival in presence of BPLP NPs (Zhang et al., 2013; Xie et al., 2014). In our study, BPLPL-based NPs showed similar cytocompatibility profile as seen for PLGA NPs with minimal cytotoxicity on HUVECs. Similar to our observation, Hu et al. (2016) also reported comparable in vitro cytotoxicity by mesenchymal stem cells as well as in vivo foreign body response toward BPLPL and PLGA materials. This suggests that inclusion of BPLP into commonly used polymers such as PLGA did not significantly affect the cell survival while the newly synthesized polymers can still inherit the florescent properties from BPLP, which could be possibly utilized for theranostic applications in CVD treatment.

Next, the proper functioning of endothelial cells in presence of BPLPL-based NPs in terms of nitric oxide production was evaluated. Nitric oxide (NO) is an important signaling molecule released by endothelial cells to regulate vascular inflammation, platelet function, angiogenesis and protection from ischemia reperfusion injury. Any dysregulation of NO production due to NOS uncoupling is known to cause cardiovascular diseases (e.g., atherosclerosis, diabetes, and hypertension) (Le et al., 2017). Inorganic nanoparticles including fluorescent silica NPs, superparamagnetic iron oxide NPs, titanium dioxide NPs generally investigated to be utilized for bioimaging applications demonstrated to induce EC toxicity and dysfunction with impaired NO production (Montiel-Dávalos et al., 2012; Astanina et al., 2014; Cao, 2018). When compared to these NP types, BPLPL-PLGA NPs could be a better alternative since it demonstrated to be inert with no effect on normal cell activities. However, this is not the case with BPLPL-PLLA NPs, nitric oxide production by endothelial cells was significantly reduced when compared to cells exposed to PLGA NPs. The mechanism that influenced the cellular behavior in presence of BPLPL-PLLA NPs is not clear. However, it is plausible that the BPLPL-PLLA nanoparticles may have upregulated oxidative stress within the cells that can activate autophagy and eventually lead to endothelial dysfunction via the PI3K/Akt/mTOR pathway as seen for silica NPs (Duan et al., 2014). Similar to our observation, Wang et al. (2014) also noted that exposure of PLLA particles to human coronary artery endothelial cells decreased their NO production, and induce inflammatory adhesion molecule expression such as ICAM-1 and VCAM-1, which might facilitate immune cell adhesion and recruitment. Furthermore, several studies reported that stents coated with PLLA impairs endothelial cell functions and impaired their recovery on the luminal side of stents that promoted in stent late thrombosis (Liu and Ma, 2010; Xu et al., 2011).

To investigate the utilization of BPLPL-based NPs as an imaging probe to track HUVECs, we incubated BPLPL-based NPs with vascular endothelial cells (HUVECs) over time. Dosedependent uptake of NPs was observed for all NP formulations. Fluorescence images demonstrated the internalization of BPLPLbased nanoparticles by endothelial cells and their subsequent localization in the cytoplasmic region of cells after a 4-h incubation. Our results agreed with previous reports from other groups that tested various nanoparticle formulations on a different cell line. For instance, Menon et al. (2014) demonstrated increasing uptake of PLGA nanoparticles by Type I alveolar epithelial cells up to 1,000µg/ml. Kona et al. (2012) also observed dose-dependent uptake of GpIbα conjugated PLGA nanoparticles and unconjugated nanoparticles by human aortic endothelial cells, which were saturated at 300µg/ml. Even BPLP particles were shown to have dosedependent uptake characteristics (Wadajkar et al., 2012). In this study, they observed BPLP particles being uptake by the human dermal fibroblast without any saturation up to 500µg/ml. It was also noted that hydrophilic and hydrophobic versions of BPLP polymers impacted the amount of nanoparticles being internalized by the different cell lines, thereby exhibiting variation in NP cellular uptake. Similarly, we have observed a significant difference in BPLPL-PLLA and BPLPL-PLGA50:50 based NPs uptake by endothelial cells, especially at concentrations of 250 and 500µg/ml. We speculate that such difference is due to differential composition of PLLA and PLGA50:50 in BPLPLs materials, where PLLA more hydrophobic in nature than PLGA50:50 and thereby affected the NP uptake by the endothelial cells. Cells continued to exhibit significant uptake of these nanoparticles at 1,000µg/ml, and as a result, almost similar amounts of NPs in the cells were observed at high concentrations despite of polymer types. On other hand, we did not observe cells demonstrate any dose-dependent NP uptake for BPLPL-PLLA NPs at concentrations <250µg/ml. In line with our results, It is plausible that the serum proteins in the media interact with the nanoparticles and modulate their uptake kinetics by the endothelial cells at low concentrations (Lesniak et al., 2010; Pelaz et al., 2015). Also, at these small concentrations, the measured levels of fluorescence intensity produced by NPs in the cells may be more difficult to discriminate.

In addition to the intrinsic fluorescence property of BPLPLs, we investigated whether the citric acid composition endows them with hemocompatibility suitable for blood contacting applications as previously seen for poly(diol-citrate) (POC) prepared from citric acid and 1,8-octanediol (Yang et al., 2004; Motlagh et al., 2007; Tran et al., 2009, 2015). First, in vitro hemostatic properties of BPLPL-based NPs were evaluated by whole blood clotting experiment. At various time points, absorbance of RBCs that were not trapped in clots were determined at 540 nm. Higher BCI values represent reduced blood clotting kinetics, and we have observed blood treated with BPLPL-based NPs did not exhibit a different rate of clotting when compared either with untreated blood or blood incubated with PLGA NPs. Second, hemolytic results of BPLPL-based NPs demonstrate them to be non-hemolytic material that is safe to be utilized for drug delivery applications without causing any adverse effects. Lastly, platelet behaviors toward BPLPLbased materials indicates that BPLPL-PLGA consisted of better or comparable platelet attachment and activation as seen in those of PLGA surfaces; whereas, higher number of platelets adhered and expressed GPIIb/IIIa markers on BPLPL-PLLA surfaces. Many studies previously observed that PLLA in its unmodified form or without incorporation of therapeutic agents induce increased inflammatory responses mainly due to its hydrophobic nature (Nguyen et al., 2003; Meng et al., 2004; Okamura et al., 2011; Rudolph et al., 2015). Our speculation is that since incorporating BPLPs into PLLA shown to increase the wettability of the polymer (Hu et al., 2016); the effect we have observed for platelets to BPLPL-PLLA could be minimal than its unmodified form of PLLA, which must be investigated further.

# CONCLUSION

We have formulated three different photoluminescent polylactones based NPs, and characterized their physical and chemical properties, protein encapsulation, in vitro hemocompatibility, cytocompatibility and particle uptake. Among these formulations, BPLPL-PLGA NPs exhibited stability in physiological conditions, bi-phasic release kinetics, excellent cytocompatibility with no negative influence on cellular functions, optimal uptake characteristics, and hemocompatibility similar to PLGA nanoparticles. Most importantly, BPLPL-based NPs showed intrinsic fluorescence capability inherited from the precursor BPLP. In short, herein we have demonstrated that BPLPL-based NPs are safe, biocompatible material with imaging capability that can be potentially used to fabricate targeted, therapeutic loaded nanocarriers for theranostic applications or utilized as an imaging agent to tag transplanted cells.

#### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

#### AUTHOR CONTRIBUTIONS

AK, KN, and JY contributed conception and design of the study. AK performed the experiments, data collection, statistical analysis, and wrote the manuscript. NP conducted TEM imaging of nanoparticles whereas DS synthesized the BPLP based

#### REFERENCES

polylactones. All authors contributed to manuscript revision, read, and approved the submitted version.

#### FUNDING

This project was supported in part by the National Institutes of Health Awards (T32 HL134613, F31 HL146118, and R21 EB024829). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

**Conflict of Interest:** JY and The Pennsylvania State University have a financial interest in Aleo BME, Inc. and Acuitive Technologies, Inc. These interests have been reviewed by the University's Institutional and Individual Conflict of Interest Committees and are currently being managed by the University.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kuriakose, Pandey, Shan, Banerjee, Yang and Nguyen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Synergistic Effect of Nanocrystals Combined With Ultrasound in the Generation of Reactive Oxygen Species for Biomedical Applications

#### Edited by:

*Michael Ming-Yuan Wei, Texas Commission on Environmental Quality, United States*

#### Reviewed by:

*Ming Xi Wan, Xi'an Jiaotong University, China Juncai Xu, Case Western Reserve University, United States*

\*Correspondence:

*Valentina Cauda<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

Received: *01 August 2019* Accepted: *14 November 2019* Published: *26 November 2019*

#### Citation:

*Vighetto V, Ancona A, Racca L, Limongi T, Troia A, Canavese G and Cauda V (2019) The Synergistic Effect of Nanocrystals Combined With Ultrasound in the Generation of Reactive Oxygen Species for Biomedical Applications. Front. Bioeng. Biotechnol. 7:374. doi: 10.3389/fbioe.2019.00374* Veronica Vighetto1†, Andrea Ancona1†, Luisa Racca1 , Tania Limongi 1 , Adriano Troia2 , Giancarlo Canavese1 and Valentina Cauda1 \*

*1 Department of Applied Science and Technology, Politecnico di Torino, Turin, Italy, 2 Ultrasounds and Chemistry Lab, Advanced Metrology for Quality of Life, Istituto Nazionale di Ricerca Metrologica, Turin, Italy*

Reactive oxygen species (ROS) effects on living cells and tissues is multifaceted and their level or dose can considerably affect cell proliferation and viability. It is therefore necessary understand their role also designing ways able to regulate their amount inside cells, i.e., using engineered nanomaterials with either antioxidant properties or, for cancer therapy applications, capable to induce oxidative stress and cell death, through tunable ROS production. In this paper, we report on the use of single-crystalline zinc oxide (ZnO) round-shaped nanoparticles, yet ZnO nanocrystals (NCs) functionalized with amino-propyl groups (ZnO-NH2 NCs), combined with pulsed ultrasound (US). We show the synergistic effects produced by NC-assisted US which are able to produce different amount of ROS, as a result of inertial cavitation under the pulsed US exposure. Using Passive Cavitation Detection (PCD) and Electron Paramagnetic Resonance (EPR) spectroscopy, we systematically study which are the key parameters, monitoring, and influencing the amount of generated ROS measuring their concentration in water media and comparing all the results with pure water batches. We thus propose a ROS generation mechanism based on the selective application of US to the ZnO nanocrystals in water solutions. Ultrasound B-mode imaging is also applied, proving in respect to pure water, the enhanced ecographic signal generation of the aqueous solution containing ZnO-NH2 NCs when exposed to pulsed ultrasound. Furthermore, to evaluate the applicability of ZnO-NH2 NCs in the biomedical field, the ROS generation is studied by interposing different tissue mimicking materials, like phantoms and *ex vivo* tissues, between the US transducer and the sample well. As a whole, we clearly proof the enhanced capability to produce ROS and to control their amount when using ZnO-NH2 NCs in combination with pulsed ultrasound anticipating their applicability in the fields of biology and health care.

Keywords: zinc oxide, nanocrystals, ultrasound, cavitation, reactive oxygen species, contrast agent

# INTRODUCTION

Reactive Oxygen Species (ROS) are the result of partial reduction of molecular oxygen (O2) (Dabrowski, 2017). The hydroxyl radical (HO·) is one of the strongest radicals ever described (Dabrowski, 2017) and it possesses the highest reduction potential of all the ROS that are physiologically relevant: due to its nature it can react with a large variety of different type of biological molecule (Fu et al., 2014).

Nevertheless, oxygen is the fundamental element needed for the normal metabolic activity of every aerobic organism, and so ROS are inevitably produced inside living organisms, as cells. ROS are normally involved in different cell functions as signaling system, induction of mitogenic response, and mitochondria activity (Fu et al., 2014). Nonetheless, the survival of cells is related to the ability of maintaining the redox homeostasis (Dabrowski, 2017) during all this processes. An instability in this equilibrium results in a variety of possible different diseases. When chronic low levels of ROS occurs in a biological living system indeed, it has been demonstrated that gene mutation and malignant cell transformation can appear, or a large variety of vascular diseases can be promoted (Lau et al., 2008). In addition, Shafique et al. (2013) established that the increase in ROS levels can have a protective role in endothelial homeostasis, improving the vascular function in patients affected by cardio vascular disease (CDV). It is also been proven that the ROS generation achieved by the external activation of membrane-bound NADPH oxidase can induce angiogenesis and other essential functions of endothelial cells, such as hemostasis (Kim et al., 2017; Aldosari et al., 2018). The activation of angiogenesis caused by an increase in ROS production to restore ROS physiological levels, can be beneficial not only for CVD, as occurs after ischemia (Urao et al., 2008), but can also contribute to wound healing (Osumi et al., 2017). On the other hand, an excessive production of ROS leads to a disequilibrium redox state, where the antioxidant defenses of the cell has been overcome, being responsible for damaging cellular components, as lipids, proteins and DNA. Acute high levels of ROS cause the activation of different signal pathways, involving cytokines, transcription factors, and mediators, responsible for cell death, causing ROS-mediated apoptosis or necrosis (Dabrowski, 2017). These effects generated by cellular oxidative stress can be exploited for cancer therapeutic applications (Pelicano et al., 2004; Nogueira and Hay, 2013; Tong et al., 2015).

Therefore, it is clear that the ability to regulate the amount of ROS generated inside cells plays a fundamental role in the survival or death of cells. During last years thus different ways to produce ROS in a controlled manner were investigated. Nanomaterials (NM) are largely studied with the aim to apply them in biomedical field, and one of the principal mechanisms of nanotoxicity is the production of oxidative stress due to ROS generation. It has also been demonstrated that the level of generated ROS is dependent on the physical and chemical properties of the considered engineered nanomaterial, as size, surface to volume ratio, and surface reactivity (Gonzalez et al., 2008; Abdal Dayem et al., 2017). Carlson et al. (2008) measured the amount of ROS produced in cells when Ag nanoparticles with different dimensions were present: 10-fold increase of ROS levels was measured in cells exposed to the smallest dimension nanoparticles. In the same work, it has been assessed that not all nanomaterials with equal dimensions can produce the same amount of ROS, supporting the idea that the ROS generation from NM depends also on their chemical nature.

Oxidative stress can also be achieved by an external activation of NMs to generate ROS, leading to tumor cell death under specific conditions. An example of this mechanism is photodynamic therapy (PDT) (Dabrowski, 2017). We have previously (Ancona et al., 2018) proposed the use of hybrid nanoparticles, able to produce intracellular ROS only when remotely activated by UV light irradiation. The photogeneration of electrons (e−) and holes (h+) have the ability to react with the environment forming superoxide radical anions (O2−) when e − reduce oxygen molecules while hydroxyl radicals (HO·) and hydrogen peroxide (H2O2) molecules are produced when h+ oxidize water molecules. In the last two decades, the use of photosensitizer materials in PDT was largely applied to cancer therapy (Dougherty et al., 1998; Dolmans et al., 2003; Dos Santos et al., 2019), but there are some limitations, as the limited tissue penetration depth of UV light used to excite the photosensitizer, that confine the application of PDT to treat superficial tumors (Dabrowski, 2017).

Ultrasound (US) is another external stimulus investigated to activate the production of ROS and sonodynamic therapy (SDT) is recently emerged as an alternative to PDT due to the higher penetration depth of ultrasound with respect to UV light (McHale et al., 2016). Additionally, under ultrasound excitation, cavitation bubbles are generated and their violent oscillation and collapse let them act as nano-chemical reactors, leading to the formation of ROS in water media. The compounds that promote ROS formation, chemically reacting or introducing a larger amount of bubbles, are named sonosensitizers (Yasuda et al., 2015). Most of the sonosensitizers, such as porphyrins, are characterized by an easy aggregation in physiological environment due to their hydrophobic nature, decreasing the therapy effectiveness, by an intrinsic toxicity and by minor selectivity to cancer tissue (Canavese et al., 2018). The effectiveness of SDT is related to the ability of efficiently generating ROS, without major drawbacks related to the nature of the implied sonosensitizer material.

A similar and most conventional technique, using photosensitizers to produce ROS is the PDT. In this treatment UV light at a specific wavelength excites the photosensitizer molecules to obtain different species of ROS and subsequent cancer cell death. PDT has been employed with promising results for the treatment of bladder, esophagus, skin, and others cancers, and is at the stage of clinical evaluation (van Straten et al., 2017). A possibility, is to combine the use of ZnO with UV in a novel PDT approach: ZnO nanoparticles have been actually employed as carrier of a photosensitizer and other chemotherapeutics (Zhang et al., 2011; Firdous, 2018) or directly as photosensitizer, as we reported recently (Ancona et al., 2018), generating superoxide, hydrogen peroxide, and hydroxyl radicals and decreasing HeLa cells viability upon irradiation. However, the main limitation of PDT is the poor tissue penetration of light, in particular the UV, that limits PDT for the treatment of superficial tumors, as melanomas. In order to overcome this drawback, a possible solution could be "tune" ZnO absorption near visible light (with increased tissue penetration rate), enveloping ZnO nanoparticles into other metals or doping them in various manners (Hu et al., 2013).

In this study, Zinc Oxide nanocrystals with a functionalized surface of aminopropyl groups (ZnO-NH2 NCs) have been proved able to produce ROS in a controlled manner, when stimulated by US generated by an already approved medical device (LipoZero G39).

Nanosized ZnO is a metal oxide well-known for its safety in biomedical fields (Racca et al., 2018). In this work, we demonstrated that our customized ZnO-NH2 NCs specific monocrystalline structure, size, shape, and functionalization, are able to generate a tunable quantity of ROS according to the intensity od administered US. More in details, the ultrasound is generated through the use of a safe medical device able to generate cavitation phenomena in human tissues. Several parameters like US output power, frequency, duty cycle, sonication time, as well as ZnO-NH2 NCs concentration in water media, were systematically examined. To push our study forward up to a possible in vivo application, it has also been verified that a larger amount of controllably cavitation and ROS generation occur also when tissue mimicking materials have been employed.

All the presented results are thus preliminary data which can potentially bring to the safe and reproducible use of nanocrystalsassisted ultrasounds for in vivo application, going from either tissue engineering proliferative effects to anticancer therapies application, thanks to the high control achieved on the amount of generated ROS.

# MATERIALS AND METHODS ZnO-NH2 NCs Synthesis and Functionalization

ZnO nanoparticles were synthesized through a microwaveassisted synthesis, as previously reported (Garino et al., 2019a). The reaction path is based on the hydrolysis of the zinc precursors (zinc acetate dihydrate) due to the presence of sodium hydroxide as the base in methanol. The as-synthesized ZnO were then functionalized with amino-propyl groups with a post-grafting approach using 3-(AminoPropyl)-TriEthoxySilane (APTES) at 10 mol% with respect to the molar amount of ZnO, as in Dumontel et al. (2017) and Garino et al. (2019a).

The obtained nanostructures are amine-functionalized zinc oxide nanocrystals (ZnO-NH2 NCs) stable colloidal suspensions in ethanol.

# ZnO-NH2 NCs Characterization

The morphological characterization of ZnO-NH2 NCs was performed by both Field Emission Scanning Electron Microscopy (FESEM, Carl Zeiss Merlin) and Transmission Electron Microscopy (TEM, FEI Tecnai operating at 200 kV) by spotting a diluted ethanolic solution of the samples (100µg/ml) on a silicon wafer for FESEM or on copper grid with 300 carbon mesh for TEM, respectively. The particles size and Z-potential value of ZnO-NH2 NCs in water suspension was determined by the Dynamic Light Scattering (DLS) technique (Zetasizer Nano ZS90, Malvern).

The crystalline structure of ZnO-NH2 NCs was analyzed by X-Ray Diffraction (XRD) with a Panalytical X'Pert diffractometer in Bragg Brentano configuration (Cu-Kα radiation, λ = 1.54 Å, 40 kV, and 30 mA).

# Evaluation of ROS Production

Ultrasound excitation was carried out with LipoZero G39 (GLOBUS) and the evaluation of ROS production was provided by Electron Paramagnetic Resonance (EPR) Spectroscopy (EMXNano X-Band spectrometer from Bruker) assisted by a spin-trapping technique. The formation of hydroxyl and superoxide anion radicals was actually detected in double distilled water using as a spin trap the 5,5-dimethyl-L-pyrroline-N-oxide (DMPO, Sigma) and each tested sample contained DMPO 10 mM. This compound is suitable for the study of ROS generation due to its capability to trap both hydroxy and superoxide anion radicals. After the ultrasound irradiation, the sample was promptly transferred into a quartz microcapillary tube and inserted in the EPR cavity. The spectra were recorded with the following measurement conditions: center field 3428 G, sweep time 160.0 s, sample g-factor 2.00000, number of scans 15. After acquisition, the spectrum was processed using the Bruker Xenon software (Bruker) for baseline correction. Analysis of recorded spectra was executed using the Bruker SpinFit software.

To perform sonication, 1 ml of sample was placed in a 24 well plate (Thermo Scientific) which was positioned in contact with LipoZero transducer through a thin layer of coupling gel (Stosswellen Gel Bestelle, ELvation Medical GmbH). Formation of hydroxyl and superoxide anion radicals was evaluated under a large range of different conditions. Samples were tested for three different sonication times (2, 5, and 10 min), five Duty Cycle conditions (10, 20, 30, 40, and 50%), three distinct working frequencies (150 KHz, 526 KHz, 1 MHz) and different output powers of the LipoZero device (0.3, 0.6, 0.9, 1.2, and 1.5 W/cm2 corresponding to 10, 20, 30, 40, and 50% of the maximum output power). In addition to these conditions, different concentrations of amino-functionalized ZnO-NH2 NCs were also tested (50, 100, and 200µg/ml) for oxygen radicals production. Temperature inside the sample well was monitored by a temperature Multilogger Thermometer 502A1 (TERSID S.r.l.).

# Needle Cavitometer Measurements

The acoustic pressure reached inside the well with LipoZero G39 at different output powers (0.6, 0.9, 1.2, and 1.5 W/cm2 ) and the acoustic cavitation activity in presence or absence of nanocrystals was monitored by recording the broad band acoustic emissions generated by collapsing bubbles by using a needle hydrophone Dapco NP 10-3 coupled to a spectrum analyzer (Agilent N9320B) and integrating the FFT area for a frequency range of 0.8–5.0 MHz. Analyses were performed at least on three spectra for each experiment.

#### B-Mode Ecographic Imaging

Ultrasound imaging was performed with a research ultrasonic scanner (Ultrasonix Sonic Touch) equipped with linear probe (L14-5/38) operating at 10 MHz in high resolution mode. It was coupled with the sample holder using ultrasound coupling gel and positioned along the axis of a single plastic well-filled with 1 ml of solution. The imaging transducer was focalized to the excitation transducer focus. Real-time videos of the system response to ultrasound irradiation were recorded and videos were analyzed using MATLAB script which calculated the relative average intensity of the bright spots in the region of interest (ROI) of each frame of the videos. Three videos were recorded for each sample.

# Tissue-Mimicking and Ultrasound Irradiation

In order to evaluate the attenuation of ultrasound effects in the presence of tissue mimicking media, different materials, as phantom and ex vivo chicken-breast tissue, were interposed between the transducer and the sample.

To conduct these tests, the ultrasound source was immersed in a plexiglass tank filled up with demineralized water and a single well, previously cut and polished from a 24 well plate (Thermo Scientific), was placed at a distance of 1 cm from the transducer surface and exposed to ultrasound, as shown in **Figure 1**.

Therefore, the measurements were performed using a tissuemimicking homogeneous phantom (based on 3% in weight of agarose and 0.4 M zinc acetate, Zn(CH3COOH)2, with an ultrasound attenuation of 0.5 dB/cm·MHz, which matches the attenuation of muscle tissue as reported in Troia et al. (2017) with a diameter of 40 mm and an ex vivo tissue (chicken breast); both materials were characterized by a thickness of 1 cm. As a reference, the effects of ultrasound irradiation on the sample were also evaluated considering only the water bath, where the system is immersed, as medium between the piezoelectric transducer and the sample well.

Ultrasound excitation was provided by LipoZero and measurements were conducted with a frequency of 1 MHz, 50% of Duty Cycle, and a power of 3 W/cm2 for 20 min. During each experiment the acoustic signal generated inside the well was recorded using a focused piezo-detector (Precision Acoustic) as a cavitometer, coupled to the Booster Amplifier (Precision Acoustic) and connected to a digital oscilloscope (TDS 2012B, Tektronix). To store the data, LabVIEW software was used and 100 µs were recorded every 2 s if the signal measured by the oscilloscope was higher than 0.001 V. Data were successively analyzed with MATLAB software. The timedomain signal was transformed in the frequency-domain by Fourier Transform and the cavitation dose was quantified with MATLAB by calculating the area subtended by the curve. The area measurements considered only values from a frequency of 2.5–12 MHz, in order to eliminate the initial 1 MHz driving signal.

At the end of sonication, EPR spectroscopy assisted by a spintrapping technique was performed as previously described. In this set of tests, EPR measurement conditions were as follows: center field 3428 G, sweep time 60.0 s, sample g-factor 2.00000, number of scans 15.

To evaluate the increase of ROS production in presence of ZnO-NH2, two conditions were tested for each tissue-mimicking material and, as a reference, for water: milliQ water with 20 mM content of DMPO and milliQ water with 20 mM content of DMPO and a concentration of NCs equal to 200 µg/ml.

SigmaPlot 14.0 software was used for all statistical analyses. Data are expressed as the mean ± standard error mean (S.E.M.). Asterisks denoting P-values (∗p < 0.05 and ∗∗p < 0.001) and sample sizes are indicated in each figure legend.

# DISCUSSION

As evidenced by FESEM and TEM analysis (**Figures 2A,B**), the ZnO-NH2 nanomaterials can be ascribed to single nanocrystalline structures, with an average diameter of 20 ± 5 nm, see also Garino et al. (2019a) for comparison. Aminefunctionalized ZnO nanocrystals have an average hydrodynamic diameter of 122 nm in their original ethanolic suspension and also in water (**Figure 2C**) and a Z-Potential value of +22 mV in double distilled water. The XRD pattern in **Figure 2D** shows the typical hexagonal wurtzitic crystalline structure of zinc oxide materials, confirming also what previously reported so far (Garino et al., 2019a,b).

The EPR spectroscopy was used to evaluate the enhancement of ROS production when ZnO-NH2 NCs are present in water (with a concentration of 200µg/ml) and according to different US power. The results are shown in **Figure 3**. Samples were irradiated with US for 10 min, at a frequency of 1 MHz and the ultrasound stimulation was pulsed, with a Duty Cycle of 10%, Pulse Repetition Frequency 1 Hz. The concentration of DMPO-OH was then evaluated since it is directly correlated to the ROS production, in particular to the hydroxyl and superoxide anions production.

The measured acoustic pressures reached inside the well at the various output powers (0.6, 0.9, 1.2, and 1.5 W/cm2 ) were between 1 and 1.5 MPa, justifying the occurrence of the inertial cavitation inside the sample well, justifying the occurrence of the inertial cavitation inside the sample well. Actually, when a lower output power was used, i.e., 0.3 W/cm2 (corresponding to 10% of the maximum output US power), also the threshold for ROS generation was not reached, meaning that inertial cavitation did not occur in the sample during the US irradiation. Otherwise, with 0.6 W/cm2 (corresponding to the 20% of the maximum output power), a small amount of hydroxyl and superoxide anions were detected, and even if the amount of DMPO-OH was greater in presence of ZnO-NH2 NCs (red bar) with respect to pure water (black bar), it can be noted that the 20% of power was not enough to obtain a statistical difference between samples. A different scenario is depicted when 0.9 and 1.2 W/cm2 were utilized. In both cases a significant difference (both with p < 0.001) between the amount of ROS produced in pure water and the one obtained in the presence of ZnO-NH2 NCs is clearly observed. The results achieved with these conditions indicate the efficacy of our ZnO-NH2 NCs to act as an ultrasound responding

pattern of the NCs.

nano-agent. It is interesting to observe that the power doses of 0.9 and 1.2 W/cm2 , corresponding to the 30 and 40% of the maximum power output of the Lipozero Transducer, were too low to generate high amount of ROS when the sonicated sample in the well was the pure water. Strikingly, both these low intensities ultrasound conditions are enough to elicit an activation of the ZnO-NH2 NCs, widely increasing the amount of ROS produced.

The last US power tested for the sonication was 1.5 W/cm2 (corresponding to the 50% of the maximum output power): the delivered intensity of US was sufficiently high to activate the inertial cavitation in the water alone, leading to a large amount of ROS produced, comparable with the one obtained in presence of ZnO-NH2 NCs.

From the results in **Figure 3**, it is assessed that 0.9 W/cm2 (30% of US power) is the optimal condition to have ZnO-NH2 NCs working as ultrasound responsive nano-agent: the significant difference (p < 0.001) in ROS production suggests that, at that power, the US irradiation was not enough intense to cause a large production of hydroxyl and superoxide anions in the

water, but it was sufficiently high to initiate the acoustic cavitation in the sample containing ZnO-NH2 NCs due to the presence of nanobubbles the NCs surface, which act as nuclei for inertial cavitation and consequently leads to a larger ROS production.

To support these results, other parameters such as ZnO-NH2 NCs concentration in water, time of US treatment, frequency of the US were tested to ensure the efficacy of the ZnO-NH2 NCs, as reported in **Figure 4**.

To assess the optimal concentration of ZnO-NH2 NCs, 50, 100, and 200µg/ml of NCs were examined (**Figure 4A**), suggesting that the highest concentration tested, as used in all the other experiments, is the optimal one. Three different treatment times were thus evaluated, 2, 5, and 10 min, keeping fixed all the other parameters (0.9 W/cm2 of power, 10% of DC, and 1 MHz of excitation frequency and 200µg/ml of ZnO-NH2 NCs in water). The insonation time of 10 min was confirmed to be the best treatment time condition to enhance the ultrasound responsive nano-agent capabilities of ZnO-NH2 NCs with respect to pure water. In **Figure 4C,** three excitation frequency were also screened: 150 KHz, 526 KHz, and 1 MHz at different US output powers, with 10% DC and 200µg/ml as NCs concentration. With the lowest frequency, a large amount of power (50% with respect to the maximum output) was needed to obtain a detectable signal with EPR instrument and DMPO spin adducts. The frequency of 1 MHz, which is the most used frequency for biomedical applications, is here confirmed to enhance the production of ROS in the presence of the ZnO-NH2 NC under ultrasound excitation.

An hypothesis for the explanation of ROS generation capabilities of ZnO-NH2 NCs is related to the NC surface: the high surface-to-volume ratio of ZnO-NH2 NCs, showing a large surface area of 60 m2 /g [as measured by Nitrogen Sorption isotherm elsewhere (Lops et al., 2019)] and the surface functionalization of amino-propyl groups are both capable to immobilize and promote the inertial cavitation of tiny gas nanobubbles under such US power conditions. As inertial cavitation is produced, ROS are generated: the EPR technique detected OH· radicals, which are one of the most reactive and potentially dangerous species of ROS.

To evaluate the role of acoustic cavitation on the generation of ROS by the ultrasound exposure of ZnO-NH2 NCs passive cavitation detection (PCD) technique was used. **Figure 5** shows the frequency spectra of the acoustic signals obtained at different ultrasound intensities with and without ZnO-NH2 NCs in solution. At low ultrasound intensities, only harmonics and subharmonics signals are present: since these signals are recorded for both water and NCs samples, they are probably due to oscillation of large gas bubbles trapped in the plastic wells of the sample holder. At increasing ultrasound intensities (above 1.2 W/cm2 ), acoustic broadband noise typical of inertial cavitation was recorded for the water solution. When ZnO-NH2 NCs were added to the solution, broadband noise signal was recorded at lower ultrasound intensities, suggesting that our NCs acted as nucleation site inducing inertial cavitation, thus decreasing the cavitation threshold. Since it has been shown both theoretically and experimentally that collapsing cavitating bubbles can generate sufficiently high temperatures and pressures able to induce generation of ROS in aqueous solution (The Acoustic Bubble, Leighton), PCD and EPR experiments together suggest that ZnO-NH2 NCs generate ROS by inducing inertial cavitation upon ultrasound exposure.

In order to further study the generation of inertial cavitation by ZnO-NH2 NCs, ultrasound B-mode imaging was used. **Figure 6A** shows the ecographic images obtained for water and ZnO-NH2 NCs containing solutions exposed to 40% intensity ultrasound. Cavitating bubble generated by ZnO-NH2 NCs led to bright spots in the solution, while in the absence of NCs the ecographic signal did not increase. **Figure 6B** shows the quantification of ecographic contrast obtained during the pulsed ultrasound exposure (170 s), as previously described in the Material and Methods section. ZnO-NH2 NCs generated higher ecographic contrast over all the sonication period compared with the water containing solution. Together these results further confirm the ability of ZnO-NH2 NCs in inducing inertial cavitation under pulsed ultrasound exposure.

ROS exert a multitude of biological effects (Lau et al., 2008; Racca et al., 2018), which also comprehend the creation of molecular damages inside cells, leading to antitumoral application (de Sá Junior et al., 2017).

In order to evaluate the future applicability of ZnO-NH2 NCs in the biomedical field, in particular to subcutaneous in vivo applications, the generation of ROS, and the effects of our NCs as ultrasound responsive nano-agent was tested in Phosphate Buffered Saline (PBS) solution, cell culture medium [Minimum Essential Medium Eagle (SIGMA) completed with 10% of Fetal Bovine Serum (FBS, SIGMA) and 1% of Penicillin-Streptomycin] and finally when different tissue mimicking materials were interposed during the insonation between the LipoZero US transducer and the sample well.

The ROS evaluation in PBS and cell culture media are reported in the **Supporting Information** and confirm the ability of our ZnO-NH2 nanocrystals to enhance inertial cavitation

and 50% of maximum available US power.

and consequently ROS production also in biological media, thus leading to applications in vitro.

The evaluation of the cavitation and ROS generation related to the interposition of tissue-mimicking materials between the ultrasound source and the samples are shown in **Figure 7**. Different materials were tested, and for all of them the amount of cavitation of water and water with the synergistic effect of ZnO-NH2 NCs (200µg/ml) were evaluated. All the measurements were performed for 20 min, with a frequency of 1 MHz, 50% of Duty Cycle, and 100% of US power available from LipoZero transducer.

**Figure 7A** exhibits the results of cavitometer measurements during 20 min of insonation, which are the average area under the Fourier Transform (FT) of each measured signal over time. These data correlate with the amount of occurred cavitation, and the S.E.M. is reported. It can be appreciated that, when

FIGURE 6 | (A) B-mode ecographic imaging of the corresponding (B) scattering signal related to bubble cavitation events obtained when 50% of maximum available US power was applied at 10% of DC, 1 MHz, and 170 s.

the ultrasound propagation medium was water, the amount of cavitation detected by the cavitometer was higher than the one measured when tissue mimicking materials were interposed. This effect can be explained considering the attenuation of power perceived inside the sample well. When the phantom and the ex vivo tissue are used, the real amount of US energy inside the well is lower, and the measured broadband noise, which correlates to cavitation, reflects this reduction. Nevertheless, a significant increase of cavitation is noticeable when ZnO-NH2 NCs are present, not only when water is the transmission medium, but also when ex vivo tissue is interposed as the propagation medium. Even if there is not a significant difference in the presence of phantom, an increase of 4% in the generated cavitation can still be noticed when ZnO-NH2 NCs are used.

At the end of 20 min of insonation, the amount of hydroxyl and superoxide anion radicals produced were evaluated for all the different conditions using a spin-trapping technique involving DMPO.

**Figure 7B** shows areas under the EPR spectrum curve, corresponding to the integrated intensity of the radical species and reflecting the concentration of DMPO-OH, index of ROS generation. As similarly reported in **Figure 7A**, the results in **Figure 7B** demonstrate that a larger amount ROS were produced when the irradiation medium is distilled water, with respect to phantom and ex vivo tissue, The results obtained lead to the conclusion that we successfully generated ROS in a controlled manner even in presence of two different tissue-mimicking materials. The data open the possibility to apply this technology in vivo for subcutaneous ROS generation using an already approved medical device.

Despite the general difference between the attenuating media, in all the cases a largest amount of ROS was detected in presence of ZnO-NH2 NCs, suggesting that our nanocrystals enhance the production of free radicals under ultrasound stimulation. This phenomenon is proved here also when tissue mimicking materials were interposed between the stimulation source and the sample, suggesting the possibility of in vivo applications.

## CONCLUSION

We report in this paper the ability of ZnO-NH2 nanocrystals in inducing inertial cavitation under pulsed ultrasound exposure. In details, it is assessed that 0.9 W/cm2 (30% of US power) is the optimal condition to have ZnO-NH2 NCs working as ultrasound responsive nano-agent and showing the significant large production of ROS, specifically of hydroxyl and superoxide anions in the water. We proposed, as mechanism of ROS generation, that this US conditions are sufficient to initiate the acoustic cavitation of tiny gas nanobubbles trapped at the ZnO-NH2 NCs surface. This inertial cavitation consequently leads to a large ROS production. Strikingly in the same insonating condition, lower cavitation and consequently largely lower amount of ROS are generated from the pure water control sample.

Ultrasound B-mode imaging was also used to confirm the generation of inertial cavitation by ZnO-NH2 NCs. An enhanced ecographic signal generation was detected when ZnO-NH2 NCs solutions were exposed to 40% intensity ultrasound with respect to pure water.

To evaluate the future applicability of ZnO-NH2 NCs in the biomedical field, the generation of ROS and the effects of NCs as ultrasound responsive nano-agent agent were tested when different tissue mimicking materials were interposed during the insonation between the US transducer and the sample well. A significant increase of cavitation is noticeable when ZnO-NH2 NCs are present, with respect to pure water, when phantoms and, in a larger amount, ex vivo tissue are interposed as the propagation medium. These measurements, together with the increased and controlled ROS production also in biological media as PBS and cell culture media (EMEM), suggest the future applicability of this technology to the in vivo setting.

All together these results proof the enhanced effects and controllability of ROS generation by ZnO-NH2 NPs assisted

#### REFERENCES

pulsed ultrasound, anticipating high potential in a wide range of biomedical/healthcare applications.

# DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

## AUTHOR CONTRIBUTIONS

VV, AA, and LR performed most of the experiments described in the manuscript. TL designed the solution and tissuemimicking measurements and assisted them. AT performed and evaluated critically the needle hydrophone and B-mode imaging characterization. GC designed the whole manuscript concept. VC supervised the whole work and was the recipient of the funding. All the authors contributed to the manuscript writing and corrections.

# FUNDING

This work had received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant Agreement No. 678151—Project Acronym TROJANANOHORSE—ERC starting Grant), and also from the Politecnico di Torino and the Moschini Spa Company through a seed funding of Proof-of-Concept Grant No. 16417.

### ACKNOWLEDGMENTS

The authors wish to thank Eng. MD Carmine Lops and Eng. MD Federico Zagallo for setting up the preliminary measurements, Eng. MD Bianca Dumontel and Dr. Nadia Garino for the nanocrystals synthesis and preliminary characterizations, and Dr. Mauro Raimondo for FESEM and TEM imaging.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2019.00374/full#supplementary-material

sonodynamic therapy against cancer. Chem. Eng. J. 340, 155–172. doi: 10.1016/j.cej.2018.01.060

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Vighetto, Ancona, Racca, Limongi, Troia, Canavese and Cauda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Gadolinium-Based Nanoparticles for Theranostic MRI-Guided Radiosensitization in Hepatocellular Carcinoma

Pengcheng Hu1†, Zhequan Fu1†, Guobing Liu1 , Hui Tan1 , Jie Xiao1 , Hongcheng Shi 1,2 \* and Dengfeng Cheng1,2 \*

*1 Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, Shanghai, China, 2 Shanghai Institute of Medical Imaging, Shanghai, China*

#### Edited by:

*Michael Ming-Yuan Wei, Texas Commission on Environmental Quality, United States*

#### Reviewed by:

*Weibo Cai, University of Wisconsin-Madison, United States Xi Yang, Fudan University, China Lihua Wang, Shanghai Advanced Research Institute (CAS), China*

#### \*Correspondence:

*Hongcheng Shi<EMAIL_ADDRESS>Dengfeng Cheng<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

Received: *12 September 2019* Accepted: *13 November 2019* Published: *27 November 2019*

#### Citation:

*Hu P, Fu Z, Liu G, Tan H, Xiao J, Shi H and Cheng D (2019) Gadolinium-Based Nanoparticles for Theranostic MRI-Guided Radiosensitization in Hepatocellular Carcinoma. Front. Bioeng. Biotechnol. 7:368. doi: 10.3389/fbioe.2019.00368* Background: Radiation therapy (RT) of hepatocellular carcinoma (HCC) is limited by low tolerance of the liver to radiation, whereas radiosensitizers are effective in reducing the required radiation dose. Multimodality gadolinium-based nanoparticles (AGuIX) are small and have enhanced permeability and retention effects; thus, they are very suitable for radiation sensitizer HCC RT. Here, we evaluated the potential value of AGuIX for theranostic MRI-radiosensitization in HCC.

Methods: The radiosensitization effects of AGuIX were evaluated via *in vitro* and *in vivo* experiments. Tumor growth, apoptosis imaging, and immunohistochemistry were performed to verify the antitumor effects of RT with AGuIX.

Results: *In vitro* evaluation of the efficacy of radiosensitivity of the AGuIX demonstrated that the presence of AGuIX significantly decreased HepG2 cell survival when combined with an X-ray beam. *In vivo* MRI imaging showed the ratio of tumor/liver concentration of the AGuIX was the highest 1 h after intravenous injection. For antitumor effects, we found that the tumor size decreased by RT-only and RT with AGuIX. The antitumor effects were more effective with high-dose AGuIX-mediated RT. Apoptosis imaging and immunohistochemistry both demonstrated that the degree of the cell apoptosis was highest with a high dose of AGuIX-mediated RT.

Conclusions: This study provides compelling data that AGuIX can facilitate theranostic MRI-radiosensitization in HCC.

Keywords: nanoparticles, AGuIX, hepatocellular carcinoma, MRI, theranostic, radiosensitization

# INTRODUCTION

Hepatocellular carcinoma (HCC), the most common liver cancer in the world, only 20–25% can be cured by surgery alone (Torre et al., 2015). Most HCC patients require comprehensive multidisciplinary treatment because they are in advanced or first diagnosed as terminal stage (Lope et al., 2012; Waller et al., 2015). Radiotherapy (RT) based on highly penetrating MeV photons (X-rays and γ-rays) is non-invasive and useful for inoperable tumors. HCC itself is a radiotherapysensitive tumor, and thus, radiotherapy plays an important role in comprehensive HCC treatment (Poon, 2011). However, radiotherapy suffers from poor tumor specificity. Photons can damage all tissues, leading to serious side effects on the normal liver tissue surrounding the tumor. These patients often have a background of cirrhosis, making them susceptible to lower doses of radiation than normal liver. The incidence of radiotherapy complications rises with increasing radiation dose in which radiation-induced liver disease (RILD) is a serious threat to patients' lives (Kalogeridi et al., 2015). Therefore, simultaneous enhancing the selectivity of tumor tissues and the bioavailability of radiation are the focus of future cancer radiotherapy.

Radiosensitizers can accumulate in the tumor tissue to increase the sensitivity of tumor cell to radiation, making tumor cells more likely to be killed by lower doses of radiation (Kwatra et al., 2013). Many drugs have been developed as HCC radiosensitizers, and the development of nanoparticles (NPs) is one important step (Kunz-Schughart et al., 2017). The use of NPs with the characteristic of preferential aggregation in tumors (even passively absorbed due to enhanced permeability and retention effects, EPR) can lead to local treatment of solid tumors (Rancoule et al., 2016). Furthermore, it has been proposed that NPs with high Z atoms are promising radiosensitizers because they may exert strong radiosensitizing effects on tumors when they are used in combination with several types of radiation of different energies (Liu et al., 2018). Gold NPs have a radiosensitizing effect on HCC (Zheng et al., 2013; Maniglio et al., 2018). However, radiotherapy in the liver region also damages normal tissues because normal liver tissue also has a high uptake of gold NPs (Balasubramanian et al., 2010). Therefore, to increase the target ratio, PEG-coated gold NPs were designed for HCC treatment. PEG-coated gold NPs increase the histocompatibility of gold NPs and prolong the circulation time in vivo. When galactose is coupled to gold NPs, it can recognize the asialoglycoprotein receptor (ASGPR) on the HCC, which improves its ability to bind to HCC and increase radiosensitization (Zhu et al., 2015). However, normal hepatocytes can also express ASGPR, and there is still a risk of RILD. Therefore, new nano-radiosensitizers are needed for HCC RT.

In 2013, Mignot et al. constructed a new type of multifunctional gadolinium nanoparticle, AGuIX, which is small (about 5 nm in diameter) and can be quickly excreted by the kidneys (Mignot et al., 2013). Due to the EPR effect, the liver background of AGuIX is much lower than that of AGuIX in most tumor tissues (Kamaly et al., 2012). With a high number of gadolinium atoms (atomic number 64), these nanoparticles can be used for enhanced magnetic resonance imaging (MRI), as well as a radiosensitization (Sancey et al., 2014).

In addition to the Compton effects and the photoelectric effect, the interaction between gadolinium atoms and X-rays also produces an Auger effect. The excited low-energy Auger electrons locally aggregate. There are more aggregation effects with more gadolinium atoms (Butterworth et al., 2012; Coulter et al., 2013). In addition, these materials have good biosafety and biocompatibility at conventional therapeutic concentrations (Morlieras et al., 2013; Bianchi et al., 2014; Bouziotis et al., 2017). Thus, AGuIX are NP radiosensitizers for integrated diagnosis and treatment of HCC.

Our previous research confirmed AGuIX uptake in the HepG2 cells and defined their biodistribution and pharmacokinetics in HepG2 tumor-bearing nude mice. This also indicated that the AGuIX accumulates in the HepG2 xenografts (Hu et al., 2017). Here, we evaluated the radiosensitization effect of AGuIX on HepG2 cells in vitro and performed MRI-guided RT using AGuIX radiosensitizer. We also conducted apoptosis Micro-SPECT/CT imaging to explore the radiosensitizing effect of AGuIX on HepG2 xenograft in vivo.

# MATERIALS AND METHODS

# AGuIX Nanoparticles

Gadolinium nanoparticles (AGuIX) were purchased from Nano-H (Lyon, France). The nanoparticles were spherical, dehydrated and sub-5 nm in diameter. Via built-in DOTA chelators the gadolinium atoms were attached to a polysiloxane shell in AGuIX nanoparticles. Rehydrated in sterile, Diethyl pyrocarbonate (DEPC)-treated water (Invitrogen, USA), AGuIX nanoparticles were stored at 4◦C according to the manufacturer's instructions.

# Cell Culture

The human HCC cell line, HepG2, was obtained from the Chinese Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). HepG2 cells were cultured (37◦C, 5% CO2) in Dulbecco's modified Eagle's medium (Gibco) supplemented with 100 IU/ml penicillin-streptomycin and 10% fetal bovine serum.

# Cell Irradiation With γ-Rays

HepG2 cells incubated in medium were first divided into four groups, and then irradiated at intensity from 1 to 6 Gy. Four different combinations were studied: A. Irradiation without AGuIX. B. AGuIX (0.5 nM) was added in the media just before the irradiation. This combination was called +IR/–incubation. C. Incubated cells with the AGuIX (0.5 nM) for 1 h and the media was changed just before the irradiation. This combination was called +IR/+washing. D. Incubated cells were with the AGuIX (0.5 nM) for 1 h and then irradiated. This combination was called +IR/– washing. The X-ray source (X-RAD 320, Precision X-Ray, North Branford, CT, USA) was used for irradiation. It was operated at 300 kV and 8 mA with a 2-mm Alfilter at a dose rate of 2.0 Gy/min.

#### Quantification of AGuIX-Mediated Cell Radiosensitization Effects via a Clonogenic Assay

The cells were washed with PBS, trypsinized, and counted after irradiation. The irradiated cells were incubated at 300 cells per plate and grow for 10 days in 10 cm dishes. They were stained with 10% ethanol dye solution and 1% crystal violet. The clones in plates were counted, and measurements were done in triplicate. Linear-quadratic (LQ) model was used for cell survival curves fitting. The radiation doses reducing cells survival rate to 37% (D1%) on radiation survival curve divided by that on corresponding curves of radiation with AGuIX was called sensitizing enhancement ratio (SER).

#### Animal Models

The protocol for animal research was approved by the medical ethics committee of Zhongshan Hospital, Fudan University. This study followed the relevant guidelines and regulations of Fudan University. Six-week-old male BALB/c athymic nude mice weighing between 16 and 18 g were obtained from Slac Biotechnology (Shanghai, China). The mice were subcutaneously injected with HepG2 cells (5 × 106 /100 µl) in the right flank.

#### In vivo MRI

To observe the distribution of nanoparticles in vivo and choose appropriate time for radiotherapy, three groups of nude mice-bearing HepG2 tumors with AGuIX (10 mg/200 µl nanoparticles injected into the tail vein) were chosen for MRI imaging. MRI scans were performed at four time points: before injection of nanoparticles, 1 h post injection (p.i.), 3 h p.i., and 6 h p.i. The MRI was a 7 T scanner (BioSpec 70/20 USR, Bruker Biospin MRI GmbH, Germany). We estimated the AGuIX's concentration in the tumor based on the signal of contrast-enhanced T1-weighted images (TR = 650 ms, TE = 700 ms, NA = 1, slice thickness = 0.7 mm, reconstruction voxel size = 256 × 256 × 700µm, FOV = 3 × 4.25 cm). According to the formula [Gd3+∞1/ST1(t)−1/ST1(t0)] where ST1(t0) is the signal strength before NPs injection and S T1(t) is the signal strength at a determined time after NPs injection, the Gd3+'s concentration was estimated based on the relaxation determined by the signal strength of contrastenhanced T1 images (Lux et al., 2011; Detappe et al., 2017).

TABLE 1 | Clonogenic assays.

*Clonogenic assays of HepG2 tumor cells post-AGuIX incubation (1 h) after RT in Ctrl, IN (–), W (*+*) and W (–) groups.*

#### Radiotherapy in vivo

To verify radiosensitization effect in vivo, Twenty four mice bearing subcutaneous right flank tumors were divided into four groups (n = 6). The diameter of the tumors were 4–6 mm. The mice in the control group and solely radio therapy group were injected with 0.5 mL of normal saline through the tail vein once a day for 2 days. The mice in the other two group were injected with 1 mg and 10 mg of AGuIX in 0.5 mL of normal saline through the tail vein, respectively. The tumor bearing mice received 6 Gy radiotherapy just 1 h after the intravenous injection on consecutive 2 days. All the mice were irradiated using an Xray source (X-RAD 320, Precision X-Ray, North Branford, CT, USA) operating at 300 kV and 8 mA with a 2-mm Al filter at a dose rate of 2.0 Gy/min with a 2 × 2 cm radiation field to cover the tumor. Other parts of the body were covered with 5 mm lead shield. Tumor growth was measured over the following days.

#### Micro-SPECT/CT Apoptosis Imaging

Twelve HepG2 tumor bearing mice were randomly divided into four groups (n = 3) and injected with 99mTc-duramycin (37 MBq/2 µg/mouse) through the tail vein. Two hours after injection, mice were anesthetized using inhalation of 2% isoflurane. Then they were scanned in a simultaneous micro-single-photon emission computed tomography/computed tomography (SPECT/CT) scanner (Bioscan, Washington DC,

*Dose enhancement effects in terms of SER for HepG2 cells incubated with 0.5 mM AGuIX in three groups. The sensitizing enhancement ratio (SER) was determined as the radiation dose reducing the survival to 37% (D0) for the radiation-only survival curve divided by that on corresponding curves of AGuIX plus radiation. SER* = *D0 (radiation-only group)/D0 (AGuIX plus radiation groups).*

USA). The SPECT/CT imaging parameters were as follow: SPECT: energy peak, 140 keV; scanning time, 35 s/projection; window width, 10%; resolution, 1 mm/pixel; and matrix, 256 × 256. CT: tube current, 0.15 mA; tube voltage, 45 keV; exposure time, 500 ms/frame, and frame resolution, 256 × 512. The HiSPECT algorithm was used for imaging reconstruction.

InVivoScope software (Version 1.43, Bioscan, Washington DC, USA) was used for imaging post-processing. Two 3D region of interest (ROI) were drawn in the region of the tumor and similar region on the contralateral muscle as background ROI. The concentration of radioactivity of each ROI (µCi/mm3 ) was determined using this software. The tumor-to-background ratio (T/B) was regarded to express tumor signal intensity with reducing inter-mice variations.

#### Immunohistochemistry

Mice were sacrificed after apoptosis imaging. Tumors were harvested from the above four groups. The harvested tumors were immersed in 4% paraformaldehyde buffered solution for 24 h. Then dehydrated and embedded in paraffin. The tumor was sectioned serially to 4 mm for TUNEL staining with a TUNEL Apoptosis Assay kit (Roche Diagnostics, Indianapolis, IN, USA) following the manufacturer's protocol. The immunostaining was evaluated by determining the histochemistry score (H-SCORE). H-SCORE = P(PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3) (Azim et al., 2015; Yeo et al., 2015).

#### Statistics

The cell growth curve was analyzed by OriginPro 8.0. Statistical analysis of image data and the tumor diameter used the Student's t test for comparisons between two groups by SPSS. In all cases, data were presented as mean ± SD (standard deviation). Statistical significance (∗ ) was set at P < 0.05.

#### RESULTS

#### Radiosensitizing Effect Exerted by Irradiated AGuIX in vitro

Significant radiosensitization effect was observed in all clonogenic assays (**Figure 1**). The cell cloning rates of the 1-6 Gy experimental groups were lower than that of the control group (P < 0.05). This shows that AGuIX has radiosensitization effects on each experimental group. The results of the dose enhancement studies are summarized in **Tables 1**, **2**. The +IR/-washing group showed the strongest radiosensitization followed by the +IR/+washing group, and finally the +IR/-incubation group. The SER values were 1.26, 1.45, and 1.52 for the cells treated with the +IR/- incubation (IN-), the +IR/+washing (W+), and the +IR/- washing (W-), respectively.

FIGURE 3 | Ratio of the MR signal intensity of Gd3+ in the tumor and liver. The T1-weighted MR images were acquired before and 1, 3, and 6 h after intravenous injection of 1 ml of AGuIX NPs ([Gd3+] = 100 mM) over three mice (1), (2), and (3). The ratios of the MR signal intensity of Gd3+ in the regions of tumor and liver were calculated.

AguIX (1 mg) AguIX (10 mg). Each group has 6 mice. The asterisk indicates

#### MRI Imaging of Tumors in vivo

AGuIXs were injected into the mice through the tail vein, and T1-weighted images were scanned at 1, 3, and 6 h (**Figure 2**). After injection of the AGuIX, we found that the tumor region (white arrow) became brighter than the background tissue and even the liver as seen by coronal scanning (**Figure 2**). With longer time, both the signal intensity of the tumor and the liver decreased gradually. The concentration of Gd3+ in the regions of interest (tumor, liver, muscle) was calculated according to the formula [Gd3+∞1/ST1(t)-1/ST1(t0)] (**Figure 3**). The Gd3+ concentration ratio of tumor/liver 1 h after intravenous injection is the highest among 1, 3, and 6 h.

#### Radiosensitization Assessment in vivo

The curve of tumor growth showed that the tumors size increased rapidly in the control group during the whole period of investigation and enlarging nearly 3.2-fold in 17 days (**Figure 4**).

panel 1 day before and 1 day after irradiation, and the three panels showed the images of mice injected by tail vein injection of normal saline 1 mg of AguIX (third panel) and 10 mg of AGuIX (fourth panel), respectively (*n* = 3). The same color scale was applied to each of the images. (B) The tumor-to-background ratio (T/B) was used to express tumor signal intensity. Independent-sample *t* test was used for statistics. The asterisk indicates statistical significance (*P* < 0.05).

statistical significance (*p* < 0.05).

In the group treated with radiotherapy alone and with AGuIX (1 mg), the tumor diameter decreased by two with a 6 Gy dose per fraction; however, it showed no statistical difference in tumor diameter throughout the period of investigation. During this process, the tumor diameter in AGuIX (–) group and AGuIX (1 mg) group decreased by ∼40 and 44%, respectively. In contrast, AGuIX (10 mg) combined with the radiation group showed that the tumors size shranked by 83%. Versus radiotherapy alone, the introduction of the AGuIX caused a significant dose enhancement effect that inhibited the growth of the tumors.

## Apoptosis Imaging and Immunohistochemistry

The radioactivity of the tumor increased progressively in the group of RT with AGuIX (10 mg) after therapy. The tumor-tobackground ratio (T/B) was 0.83 ± 0.39, 1.37 ± 0.31, 1.10 ± 0.62, and 3.87 ± 0.96 in the Control group, RT group, RT + AGuIX (1 mg) group, RT + AGuIX (10 mg) groups, respectively (n = 3, mean ± SD). The T/B of the RT + AGuIX (10 mg) was the highest among four groups and showed statistical differences vs. the other three groups (**Figure 5**). The results of H-SCORE and TUNEL staining of tumor tissues among the 4 groups were consistent with apoptosis imaging; the score of RT + AGuIX (10 mg) was also high. There was a statistical difference between the RT + AGuIX (10 mg) group and the control group (**Figure 6**).

#### DISCUSSIONS

AGuIX nanoparticles have been proven the capability of increasing the tumor cells' sensitivity to radiation therapy in a number of tumor cells (including radiation-resistant cell lines) in vitro. The SERs were observed from 1.1 to 2.5 (Sancey et al., 2014). Besides, AGuIX NPs, the first nanoparticles based on multifunctional silica with a hydrodynamic diameter under 5 nm, are sufficiently small to escape hepatic clearance and allow animal imaging by four complementary techniques (SPECT, fluorescence imaging, MRI and CT) (Lux et al., 2011). Thus, AGuIX has potential to be developed into theranostic MRIradiosensitization for HCC (Kamaly et al., 2012; Lux et al., 2015).

In order to study the radiosensitization effect of AGuIX on HCC RT, we next irradiated HepG2 cells. Our previous research showed that AGuIX can be taken up into the HepG2 cytoplasm and has a good dispersion shape in HepG2 cells, indicating that AGuIX was stable in the cells (Hu et al., 2017). The obtained of radiation dose enhancement studies in HepG2 cells (**Figure 1**) are similar to that by Porcel et al. in pancreatic cancer cells (Detappe et al., 2015). Irradiated AGuIX groups presented a stronger inhibition of cell clonogenic rates than the control group.

Radiosensitization was also observed in the groups with AGuIX regardless of whether AGuIX is incubated with cells for 1 h in our study. The mechanism of action of radiotherapy, in addition to the direct killing effect of the incident radiation beam, there may be other factors contributing as well. McMahon

et al. demonstrated Auger electrons can create a local effect and affect nanoparticles clustering, which is the main cause of the formation of reaction oxygen species (ROS) such as OH◦ , H2O2, or HOCl (McMahon et al., 2011). Also, a biological and chemical effect should be explored to account for the measured radiosensitization as well. Some of these ROS, with high chemical stabilities and a long-range action (few mm), may increase the cell death whether the nanoparticles are in the intercellular space or in cytoplasm. Therefore, the results of the study in vitro hinted that AGuIX whether in tumor intercellularly or in tumor intracellularly can both have the radiosensitization effect in vivo.

To evaluate the radiosensitization effect of AGuIX in vivo, we intravenously injected AGuIX into the HepG2 xenograft mice. For HCC, when the radiosensitizer concentration ratio of tumor/liver was the highest, it is the most suitable time point for RT to maximize therapeutic effect and minimize side effects to normal hepatic tissue. According to this hypothesis, we performed MRI on these mouse models and selected 1 h p.i. mice as the therapeutic time point for RT. The MRI data suggested that the signal intensity and contrast of the tumor lesion was significantly enhanced after 1 h AGuIX post injected. Therefore, MRI could be used for guiding the radiation process by monitoring and assisting tumor localization in real time.

The tumor growth curve in **Figure 4** showed that the introduction of the AGuIX during the radiation process result in a markedly dose increasing effect compared to radiotherapy alone, thereby inhibiting the growth of solid tumors. Tumor cell apoptosis after radiotherapy is the basis of cell death (Garcia-Barros et al., 2003; Neshastehriz et al., 2018), and apoptosis imaging in vivo can achieve early detection of tumor postradiation response, as well as accurate prediction of radiotherapy efficacy (Verheij, 2008). The 99mTc-duramycin is a relatively mature apoptosis imaging agent and has been used in apoptotic imaging of various tumors (Johnson et al., 2013; Audi et al., 2015; Elvas et al., 2016). Therefore, 99mTc-duramycin Micro-SPECT/CT imaging was performed to access the antitumor effects of imaging-guided RT with AGuIX. Combined with the apoptosis immunohistochemistry, both results demonstrated that the group of AGuIX-mediated RT showed the highest degree of cell apoptosis. These concurred with the tumor growth data. Thus, apoptosis imaging, pathology and tumor growth data suggest that the EPR-dependent accumulation of AGuIX within tumors can enhance the efficacy of radiation therapy.

#### REFERENCES

#### CONCLUSIONS

We describe a gadolinium-based nanoparticle AGuIX for MRIguided radiotherapy in HCC. The AGuIX provide better detection of tumors in imaging, and precise identified for accurate MRI-guided radiotherapy. On the other hand, the heavy elements in this novel nanoparticle can enhance radiosensitizing effect by irradiation dose deposition. The preliminary results showed that the radiosensitizing effect observed in vivo could be translated to remarkable tumor control via AGuIXradiosensitized radiotherapy. Thus, this study demonstrates that the AGuIX could be a promising theranostic nanoparticle for both MRI-guieded and enhancement of radiosensitivity. Thus, it can overcome the limitations of RT in HCC by increasing the tolerance of the liver tumor to radiation.

#### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material.

#### ETHICS STATEMENT

The animal research protocol was approved by Zhongshan Hospital medical ethics committee, Fudan University. All experiments were conducted following the relevant guidelines and regulations of Fudan University.

#### AUTHOR CONTRIBUTIONS

PH, ZF, HT, and HS conceived and designed the study. PH, GL, and JX performed the experiments. PH, HT, JX, and DC were engaged in the data interpretation and analysis. PH, ZF, and DC participated in the writing of the manuscript.

## ACKNOWLEDGMENTS

The authors are deeply grateful for the financial support from National Natural Science Foundation of China (Nos. 11875114, 81471706, and 81871407), and the Shanghai Municipal Health and Family Planning Commission Youth Fund of China (20164Y0113).

in rats. Biomaterials 31, 2034–2042. doi: 10.1016/j.biomaterials.2009. 11.079

irradiated heavy atom nanoparticles. Sci. Rep. 1:18. doi: 10.1038/srep00018

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Hu, Fu, Liu, Tan, Xiao, Shi and Cheng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Nanomedicines: A Potential Treatment for Blood Disorder Diseases

#### Nan Zhang1,2, Ming-Yuan Wei 3 and Qiang Ma1 \*

*1 Chinese Academy of Inspection and Quarantine, Beijing, China, 2 School of Life Science and Medicine, Dalian University of Technology, Panjin, China, 3 Texas Commission on Environmental Quality, Austin, TX, United States*

Blood disorder diseases (BDDs), also known as hematologic, is one of the diseases owing to hematopoietic system disorder. Chemotherapy, bone marrow transplantation, and stem cells therapy have been used to treat BDDs. However, the cure rates are still low due to the availability of the right type of bone marrow and the likelihood of recurrence and infection. With the rapid development of nanotechnology in the field of biomedicine, artificial blood or blood substitute has shown promising features for the emergency treatment of BDDs. Herein, we surveyed recent advances in the development of artificial blood components: gas carrier components (erythrocyte substitutes), immune response components (white blood cell substitutes), and hemostasis-responsive components (platelet substitutes). Platelet-inspired nanomedicines for cancer treatment were also discussed. The challenges and prospects of these treatment options in future nanomedicine development are discussed.

#### Edited by:

*Michele Iafisco, Italian National Research Council (CNR), Italy*

#### Reviewed by:

*Xin Li, Institute of Materia Medica (IMM), China Nenad L. Ignjatovic, Serbian Academy of Sciences and Arts (SASA), Serbia*

> \*Correspondence: *Qiang Ma<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology*

Received: *17 September 2019* Accepted: *13 November 2019* Published: *28 November 2019*

#### Citation:

*Zhang N, Wei M-Y and Ma Q (2019) Nanomedicines: A Potential Treatment for Blood Disorder Diseases. Front. Bioeng. Biotechnol. 7:369. doi: 10.3389/fbioe.2019.00369* Keywords: nanomedicine, blood disorder diseases, artificial blood components, treatment, emergency blood supply

# INTRODUCTION

Blood disorder diseases (BDDs), also known as hematologic, is one of the diseases owing to hematopoietic system disorder. BDDs can be broadly classified into three categories, red blood cell disease, white blood cell disease, and platelet disease. Among BDDs, iron deficiency anemia, leukemia, hemophilia, and malignant lymphoma are most widely known. Taking leukemia as an example, the early stage is extremely difficult to cure, resulting in high mortality rate. In 1948, Farber et al. realized a temporary remissions in acute leukemia, providing a promising direction for leukemia treatment (Farber and Diamond, 1948).

Researchers discovered that chemotherapy may be an effective means of treating BDDs with the high risk of causality due to immune system damage. The similar challenge exists in the bone marrow transplant treatment, which transplants normal human hematopoietic stem cells into the patient and rebuilds the patient's immune and hematopoietic system. A matching donor is rare to find, and the treatment cost is not affordable. Hematopoietic stem cells are rich in cord blood (CB), which could be considered as a bone marrow substitute. CB cells can be used for infusion therapy in special situations, because it allows a certain degree of leukocyte antigen mismatch. Patient survival rate could be improved by umbilical cord blood transplantation, such as the injection of two units of cord blood cells, or ex vitro expansion of cord blood cells (Pelosi et al., 2012). To date, this technology has been used for BDDs treatment, but some patients died of bacterial infection due to the insufficient doses (Maeda et al., 2005).

A great variety of methods for BDDs treatment have been developed in recent years. For example, as for severe thalassemia treatment, deferoxamine, or deferiprone is often used as a chelating agent to bind unstable iron to eliminate a series of effects from excessive iron content (Maggio, 2007). It was found that rituximab, an anti-CD20 monoclonal antibody, specifically depletes B cells, and reduces their damage to blood proteins or cells (Barcellini and Zanella, 2011). In addition, the modification or deletion of the telomere gene was found beneficial for the treatment of hematological diseases resulting from random or malignant transformation of hematopoietic function due to telomere shortening (Allegra et al., 2017). Genetic engineering has been attracted increasing attention for BDDs treatment. Townes et al. derived embryonic stem cells and then performed homologous recombination in vitro to change the pathogenic gene of sickle-shaped anemia, replacing the normal gene copy βββA with the disease-causing gene βββS (Townes, 2008). The diseased mice returned to normal and produced high levels of human hemoglobin. Osborn et al. efficiently induced doublestranded DNA breaks by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) method, specifically editing clinically relevant T cell receptor alpha constant (TRAC) gene targets that are highly correlated with leukemia (Osborn et al., 2016). Compared with patients with non-hematologic malignancies, hematological malignancies, such as chronic lymphocytic leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma, chimeric antigen receptors T cells (CAR T), had shown higher overall response rate and complete response rate.

Nanomedicine is a new branch of medicine that applies nanotechnology to traditional medical development. Nanomedicine focuses on designing a specific combination of nanomaterial (e.g., nanoparticle, nanocarrier, and nanovesicle) and small molecules to conceive a biocompatible carrier for delivering drugs to cancer sites effectively (Wicki et al., 2015). The effective component could be a polypeptide, a protein, a nucleotide, or a small molecule drug. Compared with traditional medicines, nanomedicine appears to be able to avoid the body's defense mechanisms, reduce the clearance rate, prevent tissue damage through regulated drug release, and improve the pharmacokinetics and biodistribution of the drug. The nanoscale component to build a nanomedicine include: polymer nanoparticles (NPs), liposomes, metal NPs, carbon nanotubes, and molecularly targeted NPs. For instance, polymer nanoparticles are often composed of hydrophobic core and hydrophilic shell, on which specific targeting moieties were coated. Quintana et al. attached folic acid, methotrexate and fluorescein to polyamidoamine dendrimer, and the targeted delivery improved the cytotoxic response of the cells to methotrexate 100-fold over free drug (Quintana et al., 2002). In addition to conventional intravenous and oral medications, lung infection disease was treated by inhalation of NPs as an adjuvant therapy (Blum et al., 2014; Jurek et al., 2014). Marrache et al. used a targeted high-density lipoprotein (HDL)-mimicking NP with contrast agents to detect vulnerable plaque and initiate preventative therapy for atherosclerosis (Marrache and Dhar, 2013).

By 2016, 14 nano-drugs for clinical treatment was approved by U.S. Food and Drug Administration (FDA). In addition, FDA approves 18 nanomedicines for cancer chemotherapy in clinical trials, 19 nanopharmaceutical formulations for being developed and clinically tested, and 15 antibacterial nano-formulations for being developed (Caster et al., 2017). The development of common clinical nano-formulations appear to reduce toxicity rather than improve efficacy (Caster et al., 2017). The cytotoxicity and the stability in complex biological environments are still under debate and limit the progresses of nano-drug development (Lim et al., 2016). On December 21, 2018, FDA approved the first treatment for rare BDDs by granting Elzonris (tagraxofusp-erzs) infusion for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in adults and in pediatric patients (2 years or above) (FDA, 2018). Tagraxofusp-erzs is a cytotoxin that a protein is comprised of human IL-3 and truncated diphtheria toxin (DT), and it targets cells that express CD123, the alpha chain of the IL-3 receptor, which is overexpressed in BPDCN (Jen et al., 2019). The drug is preferentially accepted by CD123 overexpressed cells and initiates irreversible protein synthesis to cause cell death.

Despite of the excitement of the emerging of above-mentioned nanomedicines, direct blood transfusion of whole blood is preferred, if available, to efficiently treat BDDs. However, the storage of whole blood is challenging, due to short shelf life and the risk of infection by pathogenic bacteria (Hess, 2006; Greening et al., 2010). Most of the whole blood in the blood bank are donated by volunteers. At the beginning of the twentieth century, private blood sales led to the ravage of Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) as a result of the lack of general health knowledge and safety awareness, especially in some economically backward developing countries. Taking China as an example, with the development of the country's economy, the need of blood donation, according to the National Health Service of China, has become desperately larger, but the public awareness of blood donation still falls behind. The blood banks' inventory became in shortage. Whole blood transfusion is often used for the patients after major surgeries or with BDDs, such as acute anemia. Therefore, the synthesis of blood substitutes as a supplement to the natural blood bank has become an urgent need.

Herein, we demonstrate recent advances in artificial blood components: gas carrier components (erythrocyte substitutes), immune response components (white blood cell substitutes), and hemostasis-responsive components (platelet substitutes). Moreover, the development of platelet-inspired nanomedicines was discussed. The challenges and prospects of these treatment options in future development are discussed.

 The main function of RBCs is to serve as a gas carrier component for transporting oxygen and carbon dioxide. As discussed, RBC substitutes may be an alternative solution for emergency blood transfusion for BDDs treatment. The advantages of RBC substitutes include: (a) no surface antigens; (b) more convenient to store than natural blood; and (c) function as effectively as hemoglobin.

More than three decades ago, researchers extracted RBCs from expiring human or animal blood samples, broke the cells and centrifuged, and collected cell-free hemoglobin (Kothe et al., 1985), which was considered as an early version of RBC substitute. This hemoglobin solution showed the capacity of expanding oxygen delivery (Kaplan and Murthy, 1975), whereas the short effective time led to hypoxia-induced poisoning of liver, kidney, brain, and other organs (Friedman et al., 1978, 1979; White et al., 1986).

Chemical crosslinking of hemoglobin monomers was reported to eliminate the adverse effects of hemoglobin. For example, Biro et al. tested the effects of whole blood, unmodified stromafree hemoglobin solution (SFHS), and partially cross-linked hemoglobin solution on coronary vessels in anesthetized openchest dogs (Biro et al., 1988). The results showed that unmodified SFHS significantly caused vasoconstriction compared with whole blood perfusion. Blood vessels do not shrink when SFHS is subjected to pyridyloxylation (partial crosslinking). Infusion of adenosine in the coronary artery did not improve this condition. The results indicate that unmodified hemoglobin preparations cause coronary artery contraction in dogs and have an effect on their normal blood circulation (Biro et al., 1988). When the RBC lysate is covalently linked by adenosine triphosphate (ATP) or pyridoxal phosphate and purified by agarose nucleophilic chromatography, it can cause vasoconstriction of the coronary artery. This may be caused by the covalent modification, or that nucleophilic chromatography removes membrane phospholipids and denatured protein aggregates (Vogel et al., 1987; Lang et al., 1990).

Diaspirin cross-linked hemoglobin (DCLHb) is a modified hemoglobin that the two α helix units were crosslinked while the molecule maintains a tetramer conformation (the structure is shown in **Table 1**). DCLHb had shown a strong oxygen carrying capacity. DCLHb was in the treatment of severe traumatic hemorrhagic shock: a randomized controlled efficacy trial. Early experiments showed that DCLHb was well-tolerated and had shown no obvious functional disorders or toxicity (Przybelski et al., 1999). However, Sloan et al. found that the input of DCLHb resulted in an increase in patient mortality compared to the control group (Sloan et al., 1999). A more in-depth study found that it was well-tolerated at low doses (50–200 mg/kg) (Bloomfield et al., 1996; Przybelski et al., 1996), whereas some side effects, such as yellowing skin, hemoglobinuria, and jaundice, were observed at high doses (680–1,500 mg/kg). Taken together, DCLHb could be an alternative blood supply for patients with rare blood types or for emergency usage during the shortage of blood bank inventory (Reppucci et al., 1997; Schubert et al., 2002, 2003).

Polymerized hemoglobin (PolyHeme, the structure is shown in **Table 1**) appeared to be a safer oxygen-carrying resuscitation fluid than DCLHb. It is a hemoglobin polymer complex formed by crosslinking adjacent α subunit and β subunit. In 1998, PolyHeme was first applied in trauma and emergency surgery, showing good tolerance and safety (Gould et al., 1998). Subsequent experiments showed that total hemoglobin level could be maintained by infusion of PolyHeme alone, thereby increasing the survival rate of patients with RBCs in critical situations (Gould et al., 2002). In addition to PolyHeme, researchers found that bovine aggregated hemoglobin (HBOC-201) was found to be able to keep up the hemoglobin level (Levy et al., 2002). In their work, a randomized, double-blind trial of HBOC-201 for patients undergoing heart surgery was conducted. After blood transfusion with HBOC-201, one third of patients survived without additional supply of RBCs. This indicates that HBOC-201 transfusion might be an alternative treatment for patients with moderate to severe anemia after cardiac surgery. Studies had shown that HBOC-201 is an effective oxygen carrier for most patients (Dong et al., 2006; Mackenzie et al., 2010); however, high vasoactivity-related adverse effects were found in the seniors with partial orthopedic surgery (Freilich et al., 2009).

Instead of cross-linking modification of hemoglobin, poly (ethylene glycol) (PEG) modified hemoglobin had attracted increasing attentions. For example, Nho et al. experimented with PEG-bovine hemoglobin in a low blood volume-bleeding shock model in dogs. The results showed that PEG-bHb has a longer half-life in blood vessels and no significant adverse effects compared to native hemoglobin (Nho et al., 1992). Björkholm et al. found that in a single-blind clinical trial of 12 volunteers they did not show any adverse symptoms at a dose of 50 mg/kg. When the dosage was increased to 100 mg/kg, the levels of amylase and lipase were slightly higher than normal (Bjorkholm et al., 2005). Hemospan is a PEG-modified human hemoglobin product developed by Sangart Inc and is also a widely used clinical preparation of RBCs substitutes. Eight PEG molecules were attached to the specific sites on α globulin and β globulin (Vandegriff et al., 2008), as depicted in **Table 1**. The purpose of this design is to prolong the retention time in blood vessel by increasing molecular weight, to increase the oxygen affinity, and to prevent it from early unloading (Vandegriff and Winslow, 2009). Olofsson et al. conducted a safety study in old patients undergoing selective hip arthroplasty and found that Hemospan is well-tolerated. No high frequency of adverse events from infusion to 24 h were observed along with a slight increase in liver enzymes and lipase (Olofsson et al., 2006). In a more recent study, Cooper et al. reported an PEGlated Hb engineered with tyrosine residues (Cooper et al., 2019), and it showed an increased vascular half time compared to wild type PEGlated Hb. The tyrosine residues were believed to enhance the reducibility of Hb and decrease adverse side effects, such as autooxidation.

Hemoglobin molecules were encapsulated in liposome particles (see **Table 1**), and the surface of the particles was attached with PEG-conjugated phosphatidylethanolamine (Sakai et al., 1997). Such HbV-PEG were found to be less prone to flocculation than unmodified hemoglobin vesicles. Animal experiments demonstrated that in comparison with the control group greater blood flow and faster gas exchange rates were observed for the group that had a 90% exchange

#### TABLE 1 | Nanomedicines for the gas-carrier component.

TABLE 1 | Continued

transfusion with HbV-PEG/albumin (Sakai et al., 1997). Other Hb encapsulation protocols include polymersomes, hydrogels, or porous microparticles (Baudin-Creuza et al., 2008; Piras et al., 2008; Jia et al., 2016).

Devineau et al. reported a new strategy to form Hb-based oxygen carriers by directly adsorbing Hb onto the surface of silica NPs (Devineau et al., 2018). The authors claimed that the tetrameric structure of Hb was retained for carrying oxygen and the Hb-silica NPs complex has the potential to prevent the release of free Hb, which may lead to acute renal toxicity.

In summary, the development of nanomedicine for the application of Hb-based oxygen carriers should meet the four requirements (Benitez Cardenas et al., 2017): efficient oxygen delivery, low rates of NO scavenging, resistance to oxidative degradation, and high production yields. Chemical modification (such as cross-linking) and nano-carrier assistance are two main approaches to develop Hb-based nanomedicines for carrying oxygen. Prior to clinical trials, the latter requires more characterizations on structure and stability and more efforts to ensure the Hb-loading rates (Jansman and Hosta-Rigau, 2018). While site-specific mutations of Hb molecules had been engineered to enhance oxygen affinity, the intact Hb molecules that maintain the tetrameric structure were adopted and integrated into artificial nanostructures, either being attached onto nanocarrier's surface or encapsulated inside the nanocarrier. PEGlation provide a better bio-compatibility and enhance retention time. On one hand, molecular engineering would design protective pockets or formations to prevent oxidative molecules from penetrating the Hb, which leads to the degradation of Hb. On the other hand, adding antioxidation residues, such as tyrosine, into the mutated Hb molecule could lower the chance of autooxidation of Hb.

#### Non-hemoglobin-Based Red Blood Cells Substitutes

#### Perfluorocarbon-Based Blood Substitutes

Perfluorocarbons (PFCs) emulsions are the first hemoglobinfree RBC substitutes discovered by scientists. PFCs were found to have oxygen and deoxygenation functions. Perfluoro compounds are saturated fluorocarbon molecules that have strong oxygen affinity. In early clinical practice, PFCs emulsions were administered intravenously directly or via a carrier and then left in the liver. However, some PFCs molecules hardly remain in the liver and are rapidly excreted through the breath and skin.

In 1975, Yokoyama et al. first prepared a low-toxicity perfluorodecalin emulsion (the structure is shown in **Table 1**), which eliminated the disadvantage of the previous perfluoro compound that it accumulates in the body due to poor metabolism (Yokoyama et al., 1975). Subsequently, Naito et al. (Naito and Yokoyama, 1978) prepared a mixed emulsion, namely Fluosol-DA emulsion (the structure is shown in **Table 1**), containing perfluorodecalin, perfluorotripropylamine, hydroxyethyl starch, and Krebs-Ringer bicarbonate solution. It showed a significantly increase of the amount of oxygen delivered to the patient. Although it was prone to accumulate in the liver and spleen, it could be cleared by the lungs for a certain period of time (Tremper et al., 1980, 1982; Suyama et al., 1981; Spence et al., 1994). Fluosol-DA showed a high oxygen-carrying capacity, but its effectiveness appeared to be insufficient in certain critical situations, such as anemia patients with moderate and severe blood loss (Gould et al., 1986). Because of low durability and stability in the blood vessel as well as the complexity of configuration and use, continuing development of Fluosol-DA was hindered (Riess, 1992). Oxygent, i.e., perfluorooctyl bromide, is the second generation of PFCs emulsions and had been with a wide range of applications. Oxygent has more outstanding advantages than Fluosol-DA. First, it has a stronger oxygen carrying capacity. Second, it is more convenient to prepare and use without mixing different components. Third, it has a longer shelf life, i.e., it can be stored for more than 1 year at 5–8◦C (Riess, 1992).

Keipert et al. investigated the feasibility of applying Oxygent as a blood substitute during surgery for patients under consciousness and anesthesia (Keipert, 1995). The results showed that in the state of continuous blood loss, the low level of Oxygent was able to ensure oxygen delivery, along with two side effects at high concentrations: body temperature rises (by 1–1.5◦C in 4–6 h) and platelet count reducing. The results suggested that Oxygent could be used as a blood substitute for the patient of low-blood loss surgery. In addition to being an oxygen carrier, PFCs were found to selectively increase the radiation sensitivity of tumors, which could be beneficial for an adjuvant treatment of cancer (Teicher et al., 1992; Keipert, 1995). However, flu-like side effects were documented (Lane, 1995). In 1978, Sanders et al. synthesized a new PFCs compound, FC 75 (the structure is as shown in **Table 1**). Through potential measurement and program-induced hypoxia, FC-75 supplied oxygen to the nerves at the treatment site (Sanders and Schick, 1978). Motta et al. prepared an emulsion contains 20% perfluorotributylamine (see **Table 1** for chemical structure) in 1981 for blood replacement in rabbits (Motta et al., 1981). Rabbit vital signs showed normal within 24 h of transfusion, and tissue analysis results indicated that it could act as a good oxygen carrier, despite of low toxicity.

Dodecafluoropentane (DDFPe, see **Table 1** for chemical structure) is a special oxygen carrier with high oxygen affinity and transport capacity. It showed obvious protective effects on oxygen-dependent organs, such as brain and heart (Woods et al., 2013; Strom et al., 2014). Therefore, it has been developed for the treatment of brain damage and hemorrhagic shock (Moon-Massat et al., 2014). Intravascular fluorocarbon-stabilized microbubbles were reported to protect against fatal anemia in rats, since the oxygen carried in the microbubble was supplied for the brain functions for a certain period of time (Culp et al., 2015). As a carrier, DDFPe carries oxygen but also small molecule drugs at the same time. For example, thrombolytic drug tPA in a rabbit model of ischemic stroke or ischemic stroke (Nishioka et al., 1997; Borrelli et al., 2014). After targeted binding to blood clots suspended in the flow chamber, ultrasound was used to release oxygen or drugs. DDFPe mainly accumulates in the brain, but most of them can be removed within 2 h (Arthur et al., 2017). In a more recent study, Bonanno et al. investigated the efficacy of the DDFPe as an adjunct to prehospital resuscitation (Bonanno et al., 2018). The authors found that adding DDFPe into fresh frozen platelet does not improve survival or enhance tissue oxygenation.

The study of pulmonary mechanics in ventilator-assisted PFCs treatment in animals were conducted. In 1991, Fuhrman BP et al. used a conventional ventilator to add a normal residual volume of PFCs solution (30 ml/kg) to the piglet trachea. It was found that PFCs were able to directly participate in normal gas exchange in the lungs and did not cause significant adverse effects on piglets (Fuhrman et al., 1991). Experiments in lambs with respiratory distress syndrome have also demonstrated the feasibility of this approach. When using conventional ventilators, PFCs promoted the release of oxygen and carbon dioxide, thereby increasing blood oxygen levels in lambs (Leach et al., 1993).

In the treatment of respiratory failure in rabbits caused by saline lung lavage, the incidence and mortality rates were higher when treated with positive respiratory mechanical ventilation than that with a series of perfluoroether aeration treatments (Tutuncu et al., 1993). It had been found that adequate lung gas exchange could be maintained for hours at lower airway pressures. In 1996, Gauger et al. first conducted a PFC preliminary trial of children with acute respiratory distress syndrome (Gauger et al., 1996). The tested patients were six children extracorporeal life support (ECLS). After 2–9 days of ECLS, PFCs were added into the trachea and perform PFCs-filled lung gas ventilation (partial fluid ventilation). The cumulative amount of PFCs was 45.2 ± 6.1 mL/kg (range 30– 72.5). The results showed that ECLS patients had a brief, normal respiratory exchange in the presence of PFCs. This indicates that PFCs can be safely used in the lungs of children with severe respiratory failure. Likewise, Pranikoff et al. investigated the biocompatibility of a perflubron (perfluorobromooctane) via a safety test in neonates with congenital diaphragmatics and severe respiratory failure (Pranikoff et al., 1996). PFCs was reported to enhance partial pressure of oxygen in animals with abnormal lungs (Hernan et al., 1994; Tutuncu et al., 1996). These studies have shown that PFCs can improve lung function and reduce the cost of intensive care for lung diseases.

PFC is a safe and mature oxygen carrier that can be directly chemically synthesized and has been widely used in medical treatment. As mentioned earlier, PFC molecules are generally emulsified to form particles (in micro level) and to be used as an oxygen carrier, and the emulsifier eventually decomposes in the body. The oxygen transport of PFC particles was well-known with a linear relationship between PaO2 and oxygen content, which contrasts with the sigmoidal oxygen dissociation of blood (Alam et al., 2014). Two main advantages of PFC-based oxygen transport to improve efficacy are: (a) PFC particles perfuse in the microcirculation of capillaries where no RBCs may flow; and (b) the oxygen carried by PFC particles is in dissolved state. Inspired by the PFC-based oxygen transport, PFC-base NPs were developed for multi-task nanomedicines, including bioimaging contrast agents and drug delivery carriers for diagnosis and treatment of diseases (Winter, 2014).

#### Heme-Based Blood Cell Substitute

Heme is a cyclic molecule composed of four pyrrole subunits surrounding a ferrous ion, which are most commonly recognized as one of the key components of hemoglobin. In the early studies, a ferrous porphyrin complex was embedded in a phospholipid bilayer of liposome to synthesize a liposome-heme complex (Tsuchida et al., 1988), as shown in **Table 1**. Tsuchida et al. converted the synthetic protohemoglobin into an amphiphilic heme molecule and encapsulated it in liposome. The liposome composite has a particle size of <0.1 micron. Compared to blood, the modified liposome heme has a faster oxygen reversible binding rate, a higher oxygen solubilization volume, and better stability. It also has smaller molecular weight than RBC. Artificial lung device test results indicate that the synthesized liposome heme can successfully deliver oxygen to muscle tissue (Tsuchida et al., 1988). The liposome heme molecule reversibly binds molecular oxygen in a neutral aqueous medium at 37◦C. The oxygen adduct has an oxygen binding affinity (p 1/2) of about 50 mmHg and a half-life of half a day (Tsuchida et al., 1984). In addition, the iron (II) "picket fence porphyrin" complex with one hydrophobic imidazole is also incorporated into phosphatidylcholine molecules (the component of the phospholipid bilayer). Results have shown that The liposomal iron(II) porphyrin complex can be reversible with serum in the blood of rat at 25◦C.

#### NANOMEDICINES FOR THE IMMUNE-RESPONSIVE COMPONENT

In the blood, white blood cells (WBCs) are directly related to the immune regulation. WBCs can be deployed to the invasion site of the pathogenic microorganism, surrounded by phagocytosis, or the surface antigen being presented, and the signal is transmitted to the B cell or the T cell to exert the immune regulation function of the human body. BBDs caused by imbalances in WBCs often lead to the problems with immune response.