has been proposed. They include barium sulfate, tantalum oxide, strontium carbonate, gold, etc., for X-ray/CT imaging (Wang et al., 2007; Hoekstra et al., 2014; Mastrogiacomo et al., 2017) and gadolinium, iron, perfluorocarbon-based contrast agents, etc., for MR imaging (Wichlas et al., 2012; Mastrogiacomo et al., 2017; Nakamura et al., 2018). There are certain drawbacks associated with addition of such external contrast agents. For example, it has been shown that addition of CT and MRI contrast agents affects the setting time and mechanical properties of CaP and other bone cements (Tanomaru-Filho et al., 2012; Sun et al., 2013). Similarly, addition of external magnetic contrast agents like super paramagnetic iron oxide nanoparticles (SPIONS) can cause artifacts like blooming effects (Ventura et al., 2014).

Efforts toward improving the imaging of CaPs are currently focused on refining the current diagnostic techniques for better imaging and improving the inherent contrast of CaP bioceramics. Recent advancements in MRI are an example of the former approach. The development of ultra-short echo time and zero echo time techniques has made high-resolution bone imaging possible (Ventura et al., 2013; Dou et al., 2018). HA and other CaPs are highly amenable to functional modifications via ionic substitutions. Hence, toward the latter approach, contrastenhanced CaPs have been developed by ionic substitutions (Qi et al., 2018). Since MRI and CT are two of the most common methods of imaging that, when combined together, can image both soft and hard tissues, it is very prudent to develop multimodal CT/MR contrast-enhanced CaP bioceramics.

Multimodal imaging refers to combining several imaging techniques by developing multifunctional contrast agents. Due to their multiple binding sites and functional groups, CaPs, in nanoparticulate form especially, have been widely tried as multimodal contrast agents for simultaneous diagnosis and therapeutic applications (nanotheranosis) (Qi et al., 2018). CaP nanoparticles have been used as contrast agents by conjugating with or encapsulating organic fluorophores, encapsulating gold nanoclusters, doping with elements [e.g., europium (Eu3+), manganese (Mn2+), gadolinium (Gd3+), iron (Fe3+), neodymium (Nd3+), terbium (Tb3+), dysprosium (Dy3+), etc.], conjugating with quantum dots, radiolabeling with samarium (153Sm), indium (111In), lutherium (177Lu), technetium (99mTc), etc., for imaging modalities including fluorescence, CT, MR, and nuclear imaging (Qi et al., 2018). HA nanoparticles exhibiting paramagnetism, near-infrared (NIR)

Kalidoss et al. Theranostic Calcium Phosphate Nanoparticles

fluorescence, and X-ray absorption by Eu3+ and Gd3+ doping have been developed by Ashokan et al. (2010). They had also developed CaP nanoparticles doped with both indocyanine green (ICG) and gadolinium (Gd3+), and labeled with 99mTc-MDP for combined optical, magnetic, and nuclear imaging (Ashokan et al., 2013). The synthesis of Gd3+-doped HA labeled with 153Sm for serving as a dual-modal probe for SPECT and MRI is another example for multimodal imaging using CaPs (Liu et al., 2014). Modified CaPs with therapeutic agents can be used to achieve disease diagnosis by enhancing the imaging contrasts combined with chemotherapy/gene therapy/hyperthermia therapy (HTT)/photodynamic therapy (PDT)/radiation therapy (RT), etc. (Qi et al., 2018). For example, Eu3+/Gd3+ dual-doped HA have been used for simultaneous photoluminescent and magnetic imaging along with drug delivery application (Chen et al., 2011). Folic-acidfunctionalized, Gd3+-doped HA that functions as a theranostic system with MR imaging and production of HA-159Gd-32P radioisotope by neutron activation for active targeting of osteosarcomas has been developed (Cipreste et al., 2016). Similarly, HA doped with both Fe2+ and Fe3+ ions has been developed for theranostic applications (Tampieri et al., 2012). The Fe-doped HA exhibited intrinsic magnetization and can be used for multiple applications including hyperthermia treatment as well to develop new magnetic ceramics for enhanced bone regeneration. Tseng et al. (2014) have reported that 111In-labeled lipid CaP nanoparticles preferentially accumulated in the lymph nodes via lymphatic drainage, which were used for SPECT/CT imaging-guided lymphatic gene delivery.

In the current work, a multimodal CaP drug delivery system with intrinsic antibacterial activity and enhanced contrast for CT/MR imaging modalities has been attempted by ion substitution. The CDHA of Ca/P ratio 1.61 was chosen for triple ion substitutions based on our previous results where it was found to be superior to HA as a drug-releasing resorbable bone substitute for the prevention and treatment of bone infections (Madhumathi and Sampath Kumar, 2014). Our preliminary work has shown that silver and gadolinium substitution into the HA crystal structure has resulted in significant CT and T1 MR

contrast (Madhumathi et al., 2014). Iron (Fe3+) with its large magnetic moments is used as a contrast agent in T2 relaxation mode of MRI (Chandra et al., 2012). Hence, the substitution of Fe along with Ag and Gd in CDHA for the additional T2 MR contrast will be studied. The contrast enhancement provided by the triple-ion-substituted CDHA nanoparticles will be characterized through X-ray, CT imaging, and MR imaging modes. In addition, the in vitro tetracycline loading and release profile, antibacterial activity, and biocompatibility of the substituted CDHA nanoparticles will also be evaluated.

# MATERIALS AND METHODS

# Materials

Analytical grade calcium nitrate Ca(NO3)2·4H2O, diammonium hydrogen phosphate (NH4)2HPO4, silver nitrate Ag(NO3), gadolinium oxide Gd2O3, ferric nitrate nonahydrate Fe(NO3)3·9H2O, and ammonia (30% GR) were purchased from MERCK, India. The drug tetracycline was purchased from Sigma Aldrich, India.

# Synthesis of Pure CDHA Nanocarriers

Pure CDHA of Ca/P ratio 1.61 was prepared as reported earlier through a microwave-accelerated wet chemical reaction using Ca(NO3)2·4H2O and (NH4)2HPO4 as precursors (Siddharthan et al., 2004). The reaction was carried out at pH 10 using ammonia with stirring. The precipitate formed was subjected to microwave irradiation in a microwave oven (BPL India, 2.45 GHz, 800 W power) for 20 min, after which it was washed with distilled water and dried. The dried samples were then powdered using an agate mortar and pestle.

# Synthesis of Triple-Ion-Substituted ICDHA Nanocarriers

The triple-ion-substituted CDHA was prepared by adding Ag, Gd, and Fe precursors along with Ca(NO3)24H2O and (NH4)2HPO4 using the same microwave-accelerated wet chemical method mentioned in the section Synthesis of Pure CDHA Nanocarriers. The amount of precursors was calculated by fixing Ag+ concentration at 0.25 at.%, Fe3+ at 0.25 at.%, and Gd3+ at 0.5 at.%, restricting the total substitution to 1 at.%. The (Ca+Ag+Gd+Fe)/P ratios were maintained at 1.61. The sample was labeled as ICDHA.

# Material Characterization

The phase and crystallinity of the nanocarriers were characterized by X-ray diffraction method (XRD, Bruker D8 DISCOVER, USA) using Cu Kα radiation (λ = 1.54 Å). The diffraction patterns were recorded at a scanning rate of 1 step/s with a step size of 0.1◦ /step. The functional groups present in the CDHA and ICDHA nanocarriers were analyzed by Fourier transform infrared spectroscopy (FT-IR, Perkin-Elmer Spectrum Two, Germany) in the attenuated total internal reflectance (ATR) mode. The FT-IR spectra were collected from the range 4,000**–**510 cm−1 . The elemental composition of ICDHA was studied by scanning electron microscopy (SEM, FEI Quanta 200, Netherlands) using energy dispersive X-ray (EDS) analysis. The

particles were dispersed in distilled water and ultrasonicated for 5 min. The dispersed nanoparticles were then sprayed on a cylindrical metal stub using an adhesive carbon tape. The surface was then dried and coated using gold for 2 min to improve the conductivity of the sample. Transmission electron microscopy (TEM, Philips CM20, Netherlands) was used to identify the size and morphology of the prepared nanocarriers. For TEM analysis, the apatite particles were dispersed in ethanol and ultrasonicated for 15 min. The dispersed samples were coated on carboncoated copper grids, dried, and examined under TEM operated at 120 kV. The surface area measurements of the samples were obtained at the temperature of liquid nitrogen using the Brunauer**–**Emmett**–**Teller (BET) N2 adsorption/desorption isotherm method (Sorptomatic 1990, USA). The magnetic properties of the nanocarriers were evaluated using vibrating sample magnetometry (VSM) at room temperature in an applied magnetic field of ± 1.5 T (Lakeshore VSM 7410, USA).

# In vitro Imaging Studies

The image contrast properties of the ion-substituted samples were studied using radiography (X-ray) and CT imaging. The powder samples were weighed and compressed into pellets of equal weight (200 mg) and 1 cm diameter. The X-ray radiographs were taken in a high-frequency X-ray generator (Siemens Multiphos 15, USA) along with aluminum standards of 1**–**5 mm thickness to differentiate the contrast. For CT scan, the pellets were placed inside a phantom model and imaged using a sixslice spiral CT (Philips Ingenuity Core128, USA) in a coronal section. For MR imaging, the nanocarriers were dispersed in distilled water. The MRI studies of triple-ion-substituted CDHAs were carried out by dispersing them in distilled H2O at different concentrations (10**–**200 mM) and imaged in both T1 and T2 mode using a preclinical MR system (Bruker Biospec, France) of 4.7 T.

# Drug Loading and Release

The drug loading and release studies were carried out from the ion-substituted samples using tetracycline following the procedure described earlier (Madhumathi and Sampath Kumar, 2014). About 5 mg of the drug was dispersed in 50 ml of suitable medium like ethanol or phosphate buffer solution (PBS). The CDHA and ICDHA nanocarriers (50 mg) were added to the drugdispersed solution and placed in a constant temperature water bath at 37◦C for 24 h. After 24 h, 2 ml of supernatant was removed

for estimation of drug concentration using UV**–**Vis spectroscopy (Lambda 35, Perkin-Elmer, USA). The samples were centrifuged, filtered, and dried at room temperature for 24 h.

The amount of drug loaded onto the carriers was determined by the following equation:

$$\text{Drug loading } \%= \text{ (lc } - \text{ Fc)} \div \text{ llc } \times 100 \tag{1}$$

Where Ic and Fc are the initial and final concentration of drug in PBS.

The release study was performed by dispersing 10 mg of drug-loaded carriers in 10 ml of PBS of pH 7.4. The samples were placed in a constant temperature water bath at 37◦C. The concentration of drug released was estimated by removing 2 ml of supernatant and replaced by fresh PBS at various time intervals. The drug release profile was determined by measuring the absorbance values at different time intervals (Fc) from the initial loaded concentration (Ic). All the experiments were performed in triplicate.

# In vitro Antibacterial Studies

The drug-loaded CDHA and ICDHA samples (10 mg) were added to 9 ml of nutrient broth. Pure and ion-substituted samples without drug were taken as controls. The suspensions were then inoculated with 1 ml of Staphylococcus aureus and Escherichia coli bacterial cultures and incubated at 37◦C for 24 h with shaking. The antibacterial efficacy of the samples was determined from the optical density (OD) of the cultures at 600 nm using the following equation:

$$\begin{array}{rcl} \text{Bacteria reduction } \% &=& \left\{ 1 - \text{(sample OD \div control OD)} \right\} \\ & \times 100 \end{array} \tag{2}$$

### In vitro Biocompatibility Studies

The biocompatibility of the drug-loaded triple-ion-substituted nanoparticulate system was tested against Swiss 3T3 fibroblast cells (NCCS, Pune) by the colorimetric MTT [3-(4, 5- 181dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] assay for 48 h. The Swiss 3T3 fibroblast cells were grown to confluence with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% 100× antibiotic–antimycotic liquid and incubated at 37◦C with 5% carbon dioxide in a CO**2** incubator (Astec, Japan). The cells were then trypsinized and counted with a hemocytometer (Marienfeld, Germany). They were then diluted at 5,000 cells per well, seeded in 96-well plates, and cultured for 24 h. Ten milligrams of the samples was suspended in 1 ml of DMEM and incubated at 37◦C for 24 h. The media in the 96-well plates was then replaced with 100 µl of the supernatant from the CDHA samples and again incubated for 24 h. Twenty microliters of 5 mg/ml MTT was added to each well and incubated for 4 h. The purple formazan precipitate formed selectively by live cells was solubilized in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a multimode plate reader (EnSpire, Perkin-Elmer, Singapore). The percentage of viable cells was calculated as the percentage relative to the control (standard polystyrene tissue culture plates) using the following equation:

Cell viability % = Sample OD ÷ Control OD × 100 (3)

# Statistical Analysis

The values are expressed as mean ± SD. Statistical analysis was performed using one- and two-way ANOVA wherever applicable with a p-value < 0.05 considered statistically significant.

# RESULTS

# Material Characterization

The XRD pattern of pure CDHA and ICDHA is shown in **Figure 1**. The ion-substituted samples exhibit peaks similar to pure HA (JCPDS 09-432) and also revealed no secondary phases other than that of HA, suggesting ionic substitution in CDHA lattice. The cell parameters and cell volume calculated using "Unitcell" software and the crystallite size calculated using Scherrer's formula from XRD spectra are listed in **Table 1**. The ICDHA sample also showed larger cell parameters and a crystallite size of 49 nm when compared to pure CDHA with a crystallite size of 31 nm.

The FT-IR spectra of pure and triple-ion-substituted CDHA are shown in **Figure 2**. All the vibration bands of ion-substituted CDHA correspond to that of the characteristic bands of pure CDHA. The peaks observed include lattice OH (3,573 cm−c ); vibrational OH (634 cm−v ); phosphate bands at 470, 565, 603, 962, 1,035, and 1,090 cm−c ; carbonate (1,457 and 1,423 cm−c ); and lattice water (3,422 cm−c ). Additional HPO2− 4 peak at 874 cm−p was also observed from ion-substituted CDHA samples.

**Figure 3** shows the SEM image and corresponding EDS spectra of ICDHA. It can be seen that ICDHAs appear as micron-sized aggregates in **Figure 3A**. The EDS spectra show the presence of Ag, Gd, and Fe in the samples in addition to Ca and P. The typical TEM images of triple-ion-substituted

samples can be seen in **Figure 4**. The TEM image of ICDHA samples showed large nanoparticles with acicular morphology. The particle sizes calculated from TEM micrographs using ImageJ image analysis software are listed in **Table 1**. The surface areas of CDHA and ICDHA samples from BET studies were 58 and 45 m2 /g, respectively. **Figure 5** shows the results of VSM studies presenting the magnetization properties before and after ion substitutions in CDHA nanocarriers. It can very well be seen that the diamagnetic CDHA nanoparticles showed paramagnetic behavior on ion substitution.

# X-ray, CT, and MR Imaging Studies

The X-ray radiographical image of the pellets of ICDHA in comparison to CDHA is shown in **Figure 6A**. It can be seen that the ICDHA pellet exhibited visibly higher contrast than pure CDHA. To further quantify the contrast (grayscale value), the pellets of the samples were compared along with aluminum (Al) standards of varying thickness (1–5 mm) as can be seen in **Figure 6A**. ImageJ software was used to quantify the grayscale value. **Figure 6B** shows the grayscale value graph corresponding to the X-ray image. The graph was obtained by plotting the contrast of the pellets (x-axis) with respect to the contrast of Al standards (y-axis). The graph shows that the contrast of ICDHA is comparable to that of 2.24 mm Al thickness while that of CDHA can be compared to 1.8-mm Al thickness. The substitution of elements with high density like Ag+ (10.49 g/cm3 ), Gd3+ (7.90 g/cm3 ), and Fe3+ (7.86 g/cm3 ) greatly increased the radiopacity of ICDHA compared to CDHA. The CT scan radiograph with ICDHA and CDHA samples is shown in **Figure 7**. The contrast values in CT are quantified in Hounsfield Units (HU). While both CDHA and ICDHA showed greater contrast than distilled water (control), ICDHA (2007 HU) exhibited much superior contrast than CDHA (51 HU).

The MR contrast images of ICDHA in T1 and T2 mode are shown in **Figure 8**. As observed in **Figure 8A**, the T1 MR contrast increased for the ICDHA samples with increasing concentrations. The color bar adjacent to the MR image shows the color change from bottom to top according to increase in contrast. Significant T1 contrast was observed at a concentration of 50µg/ml and increased up to the concentration of 200µg/ml. **Figure 8B** shows the concentration vs. longitudinal relaxivity (r1) plot for ICDHA samples. The r1 was calculated according to the equation:

$$(1 \div T\_1)\_{obs} = (1 \div T\_1)\_{dia} + r1M \tag{4}$$

where (1/T1)obs is the observed relaxation rate in the presence of ICDHA, (1/T1)dia is the diamagnetic relaxation rate in the absence of nanoparticles, and M is the molar concentration (Ashokan et al., 2010). The T1 relaxivity increased with an increase in the ICDHA nanocarrier concentration. The maximum r1 observed for ICDHA nanocarriers was 0.61 mM−1 s −1 for 200 µg/ml.

**Figure 9** shows the T2 MR contrast for different concentrations of the ICDHA samples (10–200µg/ml). The T2 images were obtained by pulse sequence spin-echo technique with TR/TE = 4,000/60 ms and FOV as 8 × 8 cm2 . Mild T2 contrast was observed at 10µg/ml concentration and increased for up 75µg/ml concentration with no further increase. The contrast obtained in T2 mode was much lower than that of the T1 mode, and so the T2 relaxivity graph was not plotted.

FIGURE 8 | (A) T1-weighted MR image of ICDHA substituted samples dispersed at different concentrations in distilled water and (B) longitudinal relaxivity vs. concentration graph of ICDHA.

# Drug Loading and Release Studies

The drug loading and release studies were carried out using the tetracycline antibiotic. **Figure 10** shows the drug loading percentage of ion-substituted apatites compared with pure samples. ICDHA showed a lower antibiotic loading (∼40–55%) compared to CDHA (65–70%). This may be attributed to the lower surface area of ICDHA as well as due to the replacement of Ca2+ ions with other ions that might affect the drug binding.

The drug release profile of tetracycline from CDHA and ICDHA, however, looks similar (**Figure 11**). The ion substitutions caused a minor change in the drug release profiles. The ICDHA samples showed a burst release of tetracycline with maximum release at 24 h, and the maximum release was lower than CDHA, which can be related to the changes in particle size as well as the non-stoichiometric nature of ion-substituted apatites.

# In vitro Antibacterial Studies

The results of the antibacterial studies of ion-substituted apatites against S. aureus and E. coli are shown in **Figure 12**. Although pure CDHA was not antibacterial, the ICDHA showed antibacterial activity between 20 and 25%, which can be attributed to the presence of Ag+. The antibacterial activity further increased with tetracycline loading as expected. All tetracycline-loaded samples showed >90% activity against both bacterial strains. The ion-substituted tetracycline-loaded ICDHA showed the highest antimicrobial activity against S. aureus, which is known to cause osteomyelitis and prosthetic joint infection when introduced through trauma, surgery, direct colonization from a proximal infection, or systemic circulation (Sampath Kumar and Madhumathi, 2016a).

# In vitro Biocompatibility Studies

The MTT assay results of pure and drug-loaded ICDHA apatites on Swiss 3T3 fibroblast cells are shown in **Figure 13**. It can be seen that all samples including the antibiotic-loaded CDHA and ICDHA samples showed 80–100% cell viability on Day 1 and 75–85% cell viability on Day 2. The high cell viability in ion-substituted samples may be due to the low total ionic substitution of ∼1%.

# DISCUSSION

Regeneration and healing of bone tissue post trauma, surgery, or infection often require the implantation of a natural bone graft or synthetic bone substitute. While the ability to aid and promote new bone formation is the main emphasis in developing such

FIGURE 10 | Loading profile of tetracycline from ion-substituted CDHA (ICDHA) compared to pure CDHA (*n* = 3; data shown as mean ± SD; *p* < 0.05, one-way ANOVA).

bone substitutes, it is also desirable to focus on other beneficial properties such as drug delivery ability, antibacterial activity, and optimal image contrast. In our previous studies, nanoparticulate bone substitutes with the abovementioned properties have been developed through approaches of drug loading and/or ion substitutions (Madhumathi and Sampath Kumar, 2014; Sampath Kumar et al., 2015). It was found that the substitution of ions such as Ag +, Gd3+, etc. imparted antibacterial activity and greater image contrast in CT and T1 MR modes.

Conventional MRI contrast agents enhance contrast either in T1 mode (bright signal) or T2 mode (dark signal). Recent research in the field of MR imaging focuses on the challenge

FIGURE 12 | Antibacterial activity of pure and tetracycline-loaded CDHA and ICDHA (*n* = 3; data shown as mean ± SD; *p* < 0.05, one-way ANOVA).

to develop contrast agents that enhance both dual T1 and T2 MRI contrast. These T1/T2 contrast agents are designed for a single instrument and assumed to have advantages such as signal reliability and lack of image mismatch, which can occur when using different contrast agents or different instruments (Shin et al., 2015). An overlay of the T1 and T2 signals can help distinguish the targets from the surroundings. Dual T1/T2 imaging has been achieved through complex routes such as conjugation of T1 and T2 elements [e.g., covalently attaching Gd-DTPA with dopamine-coated iron oxide nanoparticles via isothiourea linkage, manganese (Mn)-embedded iron oxide nanoparticles, etc.] and magnetically decoupled T1–T2 dual mode contrast agents [e.g., Mn-doped iron oxide (MnFe2O4) nanoparticles as the core T2 contrast material and Gd2O(CO3)2 as the shell T1 contrast material separated by non-magnetic silica layer] (Bae et al., 2010; Choi et al., 2010; Huang et al., 2014). Since the apatitic structure allows for flexible ion substitution, we aimed to develop CDHA nanoparticles capable of simultaneous T1 and T2 MR contrast along with CT by substituting both Gd3+ and Fe3+ ions. One of the main criteria in deciding the percentage of Ag+, Gd3+, and Fe3+ ion substitutions in CDHA was biocompatibility, and the total ion substitution was restricted to 1 at.%. Previous reports have shown that the Ag+ and Gd3+ ion substitutions in HA have been found to be biocompatible at up to 0.5 and 4.4 at.%, respectively (Ramesh babu et al., 2007; Getman et al., 2010). Hence, the silver concentration was fixed at 0.25 at.%, the Gd3+ concentration was restricted to 0.50 at.% allowing Fe3+ substitution of 0.25 at.%, and the maximum combined substitution was restricted to 1 at.%.

The synthesized ICDHA samples were found to be phase pure as observed from XRD pattern and FT-IR spectra. The EDS spectra showed the presence of Ca, P, Ag, Gd, and Fe ions in the ICDHA samples. TEM image of ICDHA samples showed them to be of similar morphology but larger sized compared to CDHA. The ICDHA samples showed a greater X-ray and CT contrast than CDHA. ICDHA also showed a greater magnetic moment in VSM than pure CDHA. A dual T1/T2 imaging ability was observed with the ICDHA, although the contrast enhancement obtained in T1 mode was much higher than that in T2 mode. The relaxivity plot for the T2 mode was not obtained due to the low T2 contrast probably owing to the low at.% of Fe3+ substitution compared to Gd3+. However, a dual T1/T2 contrast using very low ionic substitution was demonstrated. Drug delivery studies using tetracycline were performed to observe any modification in the antibiotic binding and release kinetics due to ion substitution in ICDHA. The studies showed a distinct reduction in the loading and a tendency toward burst release for these samples compared to pure CDHA. While the low loading may be attributed to the lower surface area of ion**-**substituted apatites and loss of Ca2+ binding sites, the burst release in ICDHA can be attributed to the release of drugs adsorbed on the surface. The antibacterial studies on S. aureus and E. coli showed a significant antibacterial activity of ICDHA due to Ag+ even without antibiotic loading. With antibiotic binding, the antibacterial activity increased to >95%. Due to the low total ionic substitution of 1 at.%, both samples showed high biocompatibility for up to 48 h even with drug loading. Further in vivo studies can validate the above observations, suggesting that ICDHA

# REFERENCES

Ashokan, A., Gowda, G. S., Somasundaram, V. H., Bhupathi, A., Peethambaran, R., Unni, A. K. K., et al. (2013). Multifunctional calcium phosphate nano-contrast agent for combined nuclear, magnetic and near-infrared in vivo imaging. Biomaterials 34, 7143–7157. doi: 10.1016/j.biomaterials.201 3.05.077

samples can act as biodegradable theranostic nanocarriers of antibiotics with inherent CT/T1/T2 MR contrast and antibacterial activity.

# CONCLUSION

In the present study, ion**-**substituted CDHA nanoparticles with antibacterial activity and enhanced CT/T1/T2 MR image contrast for theranostic applications were developed. Ions such as Ag+, Gd3+, and Fe3+, which enhance contrast in CT, T1, and T2 MR modes, respectively, were substituted into the CDHA apatitic crystal structure. Preliminary results showed that the contrast enhancement in CT/T1/T2 MR modes was significant for a low amount of ionic substitution (0.25 at.% Ag+, 0.5 at.% Gd3+, and 0.25 at.% Fe3+). These multifunctional CDHA nanoparticles were inherently antibacterial due to Ag+ substitution and also showed predominantly burst release of tetracycline antibiotic. The ion**-**substituted CDHA nanocarriers were also biocompatible due to the total ionic substitution of 1 at.%. The antibiotic-loaded triple**-**ion**-**substituted CDHA nanoparticles with antibacterial activity and multimodal image contrast are highly suitable for clinical orthopedic applications.

# DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the supplementary files.

# AUTHOR CONTRIBUTIONS

TS and MK conceived the idea. MK performed the material synthesis, characterization, drug release, and imaging experiments. RY planned and carried out the antibacterial and biocompatibility studies under the guidance of MD.

# FUNDING

The study is funded by Department of Biotechnology, Govt. of India.

# ACKNOWLEDGMENTS

We thank the Institute Hospital, Indian Institute of Technology Madras for providing the X-ray facility. We also thank Prof. N. Chandrakumar and Dr. Abhishek Banerjee of MRI-MRS Center, Department of Chemistry, Indian Institute of Technology Madras for help with the MR imaging.

Ashokan, A., Menon, D., Nair, S., and Koyakutty, M. (2010). A molecular receptor targeted, hydroxyapatite nanocrystal based multi-modal contrast agent. Biomaterials 31, 2606–2616. doi: 10.1016/j.biomaterials.2009.11.113

Bae, K. H., Kim, Y. B., Lee, Y., Hwang, J., Park, H., and Park, T. J. (2010). Bio inspired synthesis and characterization of gadolinium labelled magnetite nanoparticles for dual contrast T1 and T2 weighted magnetic resonance imaging. Bioconjugate Chem. 21, 505–512. doi: 10.1021/bc900424u

**Conflict of Interest Statement:** 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 Kalidoss, Yunus Basha, Doble and Sampath Kumar. 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.

# At the Intersection of Biomaterials and Gene Therapy: Progress in Non-viral Delivery of Nucleic Acids

Hasan Uludag1,2,3 \*, Anyeld Ubeda2 and Aysha Ansari 1

*1 Department of Chemical and Materinals Engineering, University of Alberta, Edmonton, AB, Canada, 2 Department of Biomedical Engineering, University of Alberta, Edmonton, AB, Canada, 3 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada*

Biomaterials play a critical role in technologies intended to deliver therapeutic agents in clinical settings. Recent explosion of our understanding of how cells utilize nucleic acids has garnered excitement to develop a range of older (e.g., antisense oligonucleotides, plasmid DNA and transposons) and emerging (e.g., short interfering RNA, messenger RNA and non-coding RNAs) nucleic acid agents for therapy of a wide range of diseases. This review will summarize biomaterials-centered advances to undertake effective utilization of nucleic acids for therapeutic purposes. We first review various types of nucleic acids and their unique abilities to deliver a range of clinical outcomes. Using recent advances in T-cell based therapy as a case in point, we summarize various possibilities for utilizing biomaterials to make an impact in this exciting therapeutic intervention technology, with the belief that this modality will serve as a therapeutic paradigm for other types of cellular therapies in the near future. We subsequently focus on contributions of biomaterials in emerging nucleic acid technologies, specifically focusing on the design of intelligent nanoparticles, deployment of mRNA as an alternative to plasmid DNA, long-acting (integrating) expression systems, and *in vitro*/*in vivo* expansion of engineered T-cells. We articulate the role of biomaterials in these emerging nucleic acid technologies in order to enhance the clinical impact of nucleic acids in the near future.

#### Edited by:

*Francesca Taraballi, Houston Methodist Research Institute, United States*

#### Reviewed by:

*Masoud Mozafari, Materials and Energy Research Center, Iran Evan Alexander Scott, Northwestern University, United States*

#### \*Correspondence:

*Hasan Uludag<EMAIL_ADDRESS>
#### Specialty section:

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

Received: *20 March 2019* Accepted: *15 May 2019* Published: *04 June 2019*

#### Citation:

*Uludag H, Ubeda A and Ansari A (2019) At the Intersection of Biomaterials and Gene Therapy: Progress in Non-viral Delivery of Nucleic Acids. Front. Bioeng. Biotechnol. 7:131. doi: 10.3389/fbioe.2019.00131* Keywords: biomaterials, gene medicine, nucleic acid delivery, nanoparticle, siRNA, mRNA, pDNA delivery, T-cell therapy

# INTRODUCTION

Synthetic and naturally derived biomaterials have been firmly entrenched in technologies intended to deliver therapeutic and diagnostic agents in a clinical setting. Biomaterials typically package the agents in a form that effectively deliver them to desired sites of actions without being impeded by physiological clearance mechanisms. They could additionally provide stability to agents in the physiological milieu as well as incorporate elements that can respond to physiological stimuli to enhance the functionality of delivered agents. Their ability to incorporate therapeutic and diagnostic agents is nowhere more demanding than the attempts to employ nucleic acid-based agents, so called gene medicines. Nucleic acids provide seemingly infinite opportunities to undertake molecular therapy and remedy the abnormal physiology, and furthermore "personalize" the intervention with the knowledge of patient-specific complementary information. Ever since the recombinant technology has been advanced to produce natural and engineered proteins en mass (Gräslund et al., 2008), various possibilities with gene medicines have excited clinicians that are eager to replace the conventional small molecular drugs that are prone to nonspecific effects on a multitude of cellular systems, and face resistance once the innate physiological mechanisms are induced by the rogue cells in order to overcome the drug effects. The recent explosion of molecular understanding of the participation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) biomolecules in control of the cell physiology has further garnered excitement in the field to commercialize a range of older nucleic acids (e.g., antisense oligonucleotides, plasmid DNA, etc.) and emerging agents such as short interfering RNA (siRNA), non-coding microRNAs (miRs), and messenger RNA (mRNA). The latter has particularly garnered exuberant interest from the business community with the largest biotech US IPO of 2018 being undertaken by Moderna Therapeutics that focusses on development of mRNA therapeutics.

In this review article, we will summarize recent exciting developments in gene medicines, knowledge gaps in the literature and outline future avenues of fruitful activity that will enable biomaterials to "propel" DNA and RNA based agents into the clinical realm. The range of promising nucleic acids is initially summarized providing the reader with a glimpse of clinical possibilities with them. The potential impact of different nucleic acids is presented and their perceived advantages and limitations are summarized when deployed in a clinical setting. Using the T-cell therapy paradigm, we will explore the possible impact of biomaterials in implementing this mode of therapy as a representative case for an emerging, broad-impact technology. We anticipate similar technology platforms based on ex vivo modified/expanded cells to find clinical validation in the treatment of an increasing number of diseases. Finally, we articulate emerging areas in nucleic acid therapeutics that will be impacted by employment of biomaterials, concentrating on intelligent nanoparticles (NPs), ex vivo cell expansion, mRNA delivery, and long-term transgene expression. This review will primarily focus on (i) therapeutic (rather than diagnostic) modalities, and (ii) non-viral, biomaterials-centered methods to undertake effective delivery of nucleic acids. The authors acknowledge that exciting developments are taking place in viral design and engineering to undertake clinical therapy, but we refer the reader to other sources on recent developments on this front (Schott et al., 2016; Lundstrom, 2018).

# SPECTRUM OF NUCLEIC ACIDS FOR CLINICAL UTILITY

The crux of gene medicine relies on the ability of nucleic acids to alter the physiology of a target cell. It is critical to understand the properties and physiological functions of different nucleic acids, especially at their site of action, to select the appropriate biomaterials carrier for effective transfection (**Figure 1**). The transient nature of the functional effects achieved with most nucleic acids forces the practitioners to choose the right target for an effective therapy. Targets whose silencing temporarily halts or simply slows down the pathological changes will not be desirable; oncogenes whose silencing lead to irreversible processes such as apoptosis induction, or targets that can sensitize the cells to deadly drug action subsequently are more desirable for effective outcomes. Below we inspect various types of nucleic acids based on their ability to derive distinct types of functional outcomes.

# Transgene Expression

In the original gene therapy approach, a gene of interest was introduced into the cells to tap into the native machinery to produce the therapeutic protein, in order to replace a defective version (such as a mutated, non-functional protein) or supplement an additional capability such as morphogeninduced tissue regeneration. The use of viruses has been favored to ensure effective (increased uptake) and long-lasting (chromosomal integration) transgene expression, but using plasmid DNA (pDNA) and other naked nucleic acids eliminates several undesirable viral effects, as long as the delivery is effective. It has been possible to design tissue-specific, inducible, minimally-recognizable and "mini" pDNAs to overcome various limitations of the initial pDNA configurations. In addition to circular pDNA, it is possible to rely on other configurations of functional genes; the expression cassettes may come in various molecular weights, conformation and topologies (Sum et al., 2014). Lower molecular weight mini pDNA vectors, both linear and circular conformations, show better cytoplasmic diffusion compared to their parental plasmid precursors. Ministring DNA vectors, which are mini linear covalently closed DNA vectors, demonstrate improved cellular uptake, transfection efficiency, and target gene expression in comparison to isogenic minicircle DNA, which are mini circular covalently closed DNA vectors, of the same size and structure as the ministring DNA (Nafissi et al., 2014). Simultaneous delivery of two pDNAs is employed in the sleeping beauty (SB) transposon system, wherein one pDNA carries the SB transposase gene while the other pDNA carries the gene of interest flanked by the transposase recognizable terminal inverted repeats (TIRs). The capability of the transposon system to permanently insert transgene constructs in the host genome and relatively superior biosafety profile, makes the SB approach advantageous over non-integrating non-viral vectors and viruses, respectively (Kebriaei et al., 2017; Tipanee et al., 2017a). We (Hsu and Uludag, 2008) and others (Dhanoya et al., 2011) have previously shown that polymeric gene carriers can condense and deliver widely different DNA molecules. How cells process different DNA molecules is an understudied area with important implications in transgene expression efficiency; comparative assessment of uptake of nucleic acid complexes (Hsu and Uludag, 2008; Symens et al., 2013; Levacic et al., 2017), nuclearlocalization (Dhanoya et al., 2011), intracellular diffusion, and increased propensity for dissociation and/or endosomal release (Ribeiro et al., 2012) remains to be fully investigated

**Abbreviations:** DNA, deoxyribonucleic acid; RNA, ribonucleic acid; siRNA, small interfering RNA; shRNA, short hairpin RNA; mRNA, messenger RNA; miR, microRNA; pDNA, plasmid DNA; SB, sleeping beauty; TIR, Terminal Inverted Repeat; ASO, Antisense oligonucleotide; RNAi, RNA interference; lncRNA, Long non-coding RNAs; circRNA, circular RNA; gRNA, guide RNA; PEG, polyethyleneglycol; PEI, polyethyleneimine; mAb, monoclonal antibody; CAR, Chimeric Antigen Receptor; APC, Antigen presenting cell; GAM, Gene Activated Matrix; TAM, Transcript Activated Matrix; TCR, T-cell Receptor; NP, nanoparticle; PLGA, polylactide-co-glycolide; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat.

especially in clinically relevant cells, but the ease of industrial expansion favors pDNA of various configurations for large scale applications. To overcome any transcriptional barriers (such as nuclear targeting and recognition by transcription factors), recent attempts have focused on delivering mRNA that can remain in cytoplasm and access the translational machinery readily (see section mRNA Delivery to Replace pDNA Therapy for more details on mRNA delivery).

# Gene Silencing

In order to silence unwanted or undesirable genes, antisense oligonucleotides (ASO; 16–20 nucleotide long single-stranded DNAs) that "neutralize" and block translation of target mRNAs were initially pursued that rely on ex vivo chemical synthesis and delivery. Apart from silencing defective genes, ASOs are finding applications in restoring the correct splicing patterns of pre-mRNAs that possess aberrant sequence elements involved in splicing or aberrant splice sites, as well as in altering expression levels of splice variants to affect a change in the function of a gene. This is executed by designing ASOs complementary to specific splice sites, thus blocking spliceosome assembly at the targeted splice site, which thereby leads to a shift of the splicing machinery to another splice site. The ASOs are expected to be capable of entering the cell nucleus, which is the site for premRNA splicing. This is achieved by employing nucleotide bases, sugars and internucleotide linkages with modified chemistries (Kole and Sazani, 2001). A notable example of utilizing ASOs in this modality is the development of exon skipping therapy, wherein ASOs are used to restore the reading frame by skipping an exon or exons containing disease-driving mutations. Exon skipping therapy has been well-explored clinically in the context of Duchenne Muscular Dystrophy (Cirak et al., 2011; Goemans et al., 2016; Mendell et al., 2016; Aartsma-Rus et al., 2017), with an ASO drug receiving FDA approval in 2016 (Sarepta Therapeutics)1 .

The endogenous RNA interference (RNAi) mechanism has been adopted for therapy by silencing genes based on blockage and/or degradation of corresponding mRNAs. RNAi can be implemented with synthetic short interfering RNAs (siRNAs; 19– 27 nucleotide long double-stranded RNAs), as well as in situ production of silencer molecules (short hairpin RNAs; shRNAs) through typical pDNA-based expression vectors. While the latter relies on nuclear targeting for efficient expression, siRNAs can be delivered to the cytoplasmic space to engage the RNA-induced silencing complex (RISC) directly with minimal processing by host cells. To achieve sustained silencing of gene expression, siRNA needs to be continually supplied exogenously, or stable integration and expression is required in the case of shRNAs. The exciting possibilities with RNAi was recently (2018) confirmed with the FDA approval of the first siRNA based drug (Patisiran by Alnylam) to treat the nerve damage caused by the rare disease hereditary transthyretin-mediated amyloidosis (hATTR) in adults. To further regulate gene expression, endogenous miRs, the non-coding single stranded RNAs with 19 to 25 nucleotides and mis-matched base pairing, can be introduced into host cells either to augment or "mimic" a particular miR. In cases where the elevated miRs themselves are the cause of underlying pathophysiology and need to be down-regulated, single stranded RNA molecules with sequence complementary to a target miR, an anti-miR, could be deployed.

Gene suppression can also be executed by utilizing a subset of RNAs, called ribozymes or catalytic RNAs, that possess enzymatic action and can cleave target mRNA with high specificity to prevent protein translation (Abera et al., 2012). Besides utilizing artificially engineered ribozymes exogenously, significant efforts have been directed toward intracellular expression of these molecules. Expression cassettes with different kinds of promoters (e.g., long-acting, cell-specific, inducible, etc.) have been explored to optimize activity of the expressed ribozyme in vivo. To enhance stability and ensure proper folding into its active structure, ribozymes are expressed as part of a larger transcript, called carrier RNA, the sequence of which is carefully chosen so that the resulting transcript not only adopts a secondary structure that does not impede ribozyme activity but is also stable (Cagnon et al., 1995; Good et al., 1997; Prislei et al., 1997). Apart from downregulation of aberrant genes by transcleaving ribozymes, pathogenic genes can be repaired and/or reprogrammed by another subset called trans-splicing ribozymes. In the repair modality, mutated genes are cleaved and replaced by wild-type RNA sequences to yield properly functioning genes while maintaining the spatial and temporal gene expression patterns in cells and tissues (Byun et al., 2003; Shin et al., 2004). In malignancies and pathological tissue induction, where multiple pathways are dysregulated, repairing a single gene may not be sufficiently effective and hence require expression of multiple therapeutic genes. Trans-splicing ribozymes with reprogramming capabilities have been developed that not only remove viral transcripts and tumor-related genes but also induce cell death leading to elimination of virus-infected and cancer cells (Won and Lee, 2012; Carter et al., 2014; Kim et al., 2017). Since reprogramming genes comes with a great risk of unconstrained expression and unintended gene removal, it is paramount to introduce elements that maintain a check on the activity of transsplicing ribozymes. This has been achieved by incorporating miR target sites or hypoxia-inducible elements in ribozymes to regulate their activity in a miR expression status-dependent manner or the ambient cellular environment, respectively. The potential of trans-splicing ribozymes in gene therapy and as anti-viral and anti-cancer tools has been reviewed elsewhere (Lee et al., 2018).

# "Sponging" Nucleic Acids

Long non-coding RNAs (lncRNA) have been recently identified whose sole function seem to sequester and alleviate the effects of intracellular molecules responsible for undesirable changes. It may be possible to alter the activity of specific DNA, RNA, and protein targets by deploying lncRNAs 'sponges.' Sponging may be additionally undertaken by so called circular RNAs (circRNAs) distinct from linear lncRNAs and miRs, featuring higher cellular stability due to the absence of free ends and resistance to exonucleolytic degradation. CircRNAs may harbor one or more binding sites for a single miR or possess binding sites for multiple miRs, thereby regulating entire miR families (Panda et al., 2016; Chen et al., 2017; Hsiao et al., 2017). Besides miR sponges, circRNAs harboring a high density of binding sites for one or more RNA-binding proteins, serve as protein sponges, thereby modulating levels of target proteins which leads to changes in downstream intracellular events (Ashwal-Fluss et al., 2014; Abdelmohsen et al., 2017). Alternatively, they may operate as protein scaffolds facilitating contact between two or more proteins when they possess binding sites for enzymes and their substrates (Du et al., 2017). Their sponging capacity can be harnessed by engineering them to include combinations of miR and protein binding sites to target specific disease profiles. Since they can also serve as templates for protein expression in the presence of appropriate translation signals (Legnini et al., 2017; Pamudurti et al., 2017; Yang et al., 2017), expression cassettes

1FDA letter to Sarepta Therapeutics Inc. on eteplirsen approval. Available online at https://www.accessdata.fda.gov/drugsatfda\_docs/nda/2016/206488\_summary %20review\_Redacted.pdf

for therapeutic proteins can be incorporated into circRNAs for gene therapy. Research on the practical implementation of therapeutic circRNAs is still in its infancy, with significant room for exploratory studies to realize their therapeutic potential.

Other nucleic acids that can affect cellular events by sequestering biomolecules are DNA and RNA aptamers. They are 56–120 nucleotides long, single-stranded synthetic oligonucleotides that can bind to various targets including small organic compounds and proteins, both intracellular and extracellular, with high affinity and specificity. They can fold into three-dimensional (3D) structures for binding to their target proteins through structural recognition and inhibit their interactions with other molecules in a manner similar to protein antagonists and antibodies, thereby serving as decoys. Their ability to recognize and bind highly structured nucleic acid targets lends them a unique functionality that may be more advantageous over previously mentioned agents (i.e., ASOs, siRNAs, miRs, ribozymes etc.). While extracellular molecules can be targeted by exogenous aptamers with relative ease, in situ production of aptamers through expression vectors has been explored for more efficient targeting of intracellular molecules (aptly known as "intramers") (Chaloin et al., 2002; Choi et al., 2006; Mi et al., 2006). Many different promoter systems and expression cassettes have been designed to obtain high intracellular levels and sustained expression of aptamers. The functionality of endogenously expressed RNA aptamers can be impaired by flanking sequences in the RNA aptamer transcript, as interaction with them can hamper proper folding and render them inert (Sullenger et al., 1990; Blind et al., 1999; Martell et al., 2002). To overcome this limitation, sequences coding for ribozymes have been incorporated into the vector, which upon expression cleaves the aptamer from the nascent RNA transcript (Joshi and Prasad, 2002; Nishikawa et al., 2003). A strategy to direct intramers to extra-nuclear compartments and localize them close to their target(s) is to include nuclear export signal sequences, which enable translocation of expressed aptamers through nuclear pore complex (Grimm et al., 1997; Hamm and Fornerod, 2000). Control over intramer activity can be attained by employing a bi-aptamer construct where one aptamer serves as a sensor for the biological trigger and the other aptamer exerts inhibitory action. The expression cassette for these trigger-inducible systems include a connection sequence between the two aptamer sequences, resulting in a functional fusion product (Ausländer et al., 2011). In a separate avenue of exploration, the capacity of aptamers to bind a diverse range of targets has been extensively exploited for derivatization of NP delivery systems for site- or targetspecific delivery. The success of this strategy is evident by numerous therapeutic RNA aptamers undergoing clinical trials (Sundaram et al., 2013; Sridharan and Gogtay, 2016).

# Gene Editing

Recent advances in gene editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease is providing exciting possibilities but also raising the bar for biomaterial-mediated delivery. The CRISPR/Cas9 system requires a single guide RNA (gRNA) and the Cas9 nuclease to undertake gene editing. For practical implementation, alongside the gRNA, the Cas9 nuclease may be delivered directly as a protein, as a pDNA cassette for protein expression in host cell, or as a "translatable" mRNA (Lino et al., 2018; Wang et al., 2018). Co-delivery of different cargoes presents a great challenge in designing an optimal delivery vehicle due to differences in physical structure and chemical properties of the different types of cargo. For instance, in contrast to anionic nucleic acids, the Cas9 protein is cationic (Sun et al., 2015), so that biomaterials have to accommodate the contrasting features of the cargo during the packaging and delivery. Biomaterials capable of optimally complexing long-string like transposase mRNA will be different from carriers that optimally interact with gRNA, so that mutually compatible carriers are likely to require concerted efforts. The situation is analogous to the SB transposon system, where supplying transposase from in situ translated mRNA (preferable to avoid the risk of chromosomal integration) (Wiehe et al., 2007; Holstein et al., 2018) instead of pDNA expression cassette will require distinct optimization of the biomaterial carrier to accomodate both types of cargo. We had previously articulated on the importance of delivering multiple agents as being the preferred approach in the case of most pathophysiologies (e.g., cancers where the internal physiology is altered in several respects) (KC et al., 2017). Deploying a single agent, while convenient for pharmacological development, may not be as effective in controlling the disease in these cases. Augmenting a defective gene may need to be undertaken while suppressing other mediators or augmenting other genes and miRs, for which combinatorial delivery of different nucleic acids will be needed (KC et al., 2017). In attempts to co-deliver a pDNA and siRNA, for example, one is faced with the delivery of a long flexible DNA molecule (>3000 bp) and a short rigid RNA molecule (<30 bp). Composite materials or chemically-distinct delivery vehicles capable of self-assembling into functional structures with different nucleic acids are needed to this end. While technically challenging, undertaking combinatorial delivery may offer the advantage of enhancing the biosafety and toxicity of certain vectors given the improvements in efficacy and the need to deploy a lower dose of the therapeutic agents.

# "Hybrid" Nucleic Acids for Responsive Systems

The simplicity of the four-nucleotide chemistry and Watson-Crick base pairing provides significant room for flexibility, as a consequence of which generation of different combinations of polynucleotides has been feasible. Chimeric constructs developed by combining different types of nucleic acids allows us to benefit from their respective desirable characteristics and diverse functionalities. The targeting capability of aptamers has been harnessed by conjugating them with ASOs, siRNAs, shRNAs, and miRs for cell-specific delivery and subsequent gene silencing (Soldevilla et al., 2018). Additionally, the unique binding capability of aptamers to small molecules has been employed to generate RNA-based regulators that integrate sensing functions and endogenous gene regulation, through ligand-responsive aptamer-miR chimeras (Beisel et al., 2011). Molecular switches that turn on/off a certain function contingent upon a physiological signal has been manifested by integrating aptamers with ribozymes, known as aptazymes. It has been shown that target interaction with aptamer induces adaptive folding around the bound target leading to adoption of a distinct conformation as well as further stabilization of adjacent helical domains. This stabilization affects the conformation of the attached ribozyme leading to a switch in its activity (Famulok et al., 2007). These triggerable systems, once optimized, permit better control over relatively complicated therapeutic strategies that target regulatory networks or genome reprogramming, as reported in a study where an aptazyme was embedded within the gRNA of a CRISPR/Cas9 system (Tang et al., 2017). By inserting multiple aptamer sequences harboring specificities for different ligands, it may be possible to obtain more precise control over genome editing and subsequently over spatial and temporal gene expression patterns to rectify diseased states.

Apart from aptamers, DNA could also serve as a delivery vehicle when folded into nanostructures through the scaffolding DNA origami technique, as demonstrated by successful in vivo delivery of siRNA by a DNA tetrahedron (Lee et al., 2012). Although the added functionality imparted by hybridizing different nucleic acids has a great appeal, especially in the context of gene therapy, it is critical to ensure that integration does not hamper the biological activity of either molecule. Other practical considerations such as synthesis cost:yield ratio, benefit:cost ratio, suitability and need for the intended application should also be evaluated during design and creation of these types of hybrid constructs.

# Nucleic Acids Without Carriers

Finally, we note that numerous clinical studies are underway where new generation of nucleic acids are being deployed without the use of a carrier (**Table 1**). Presumably, the rationale behind this approach is to avoid the introduction of synthetic carrier materials which may not be degraded at times and hence may accumulate leading to adverse effects. Carrier-free delivery eliminates the process of development and optimization of a delivery vehicle, but however poses its own set of challenges. Several factors such as poor permeability to cell membranes due to their anionic nature, rapid clearance owing to their small size, and susceptibility to degradation by ubiquitous nucleases, render the nucleic acids unfavorable in their native form. To overcome these physiological barriers, chemical modifications have been incorporated in their sugar-phosphate backbones as well as sugar and base moieties. While these modifications confer desirable attributes such as enhanced stability, nuclease resistance, target binding affinity, and reduced immune stimulation, it is crucial to include them in a balanced proportion to circumvent loss of potency. Besides chemical modifications, nucleic acids are conjugated with lipid moieties or polyethylene glycol (PEG) or small cationic proteins to aid in increasing their size, traversing the complex physiological milieu, enhancing circulation half-life, and eventually potentiating their therapeutic efficacy. Practically, utilization of naked nucleic acids seems most appropriate for localized treatment strategies, as is evident from **Table 1**, where ∼60% of the indicated clinical studies employ subcutaneous, intramuscular, intravitreal or other localized routes of administration. In these modes, they are challenged with relatively less physiological hurdles to reach their site of action and carry out their activity. Accordingly, carrierfree nucleic acid therapeutics are suitable for external and/or easily accessible tissues such as ocular, epidermal, pancreatic, pulmonary, and colonic tissues. The route of administration has a significant influence on drug biodistribution, bioavailability, and eventually its therapeutic efficacy, so that initial focus on the development of nucleic acid therapeutics for ailments of the eye, skin, and muscle are understandable. Local administration also allows for implementing gene medicines by patients through eye drops and nasal sprays. However, for more deep-seated maladies, naked nucleic acids may not be satisfactory as in this case they need to be administered systemically and are required to seek out the diseased tissue in the complex in vivo environment to be effective. For this, they need to be equipped with the right elements to identify target tissues and evade degradation, while still be biologically active. Incorporating chemical modifications can adversely effect potency, making it incumbent to utilize carriers for nucleic acid therapeutics intended for these applications. While viruses are efficient and effective carriers, significant effort has been invested in developing safer, less immunogenic non-viral techniques and biomaterials for delivering nucleic acid therapeutics in hematological malignancies, as reviewed elsewhere (Ansari et al., 2017).

# TECHNOLOGY FOR CELLULAR ENGINEERING: T-CELL THERAPY AS A CASE IN POINT

The exciting developments in T-cell therapies are providing opportunities for biomaterials to implement a new type of gene medicine. T-lymphocytes are essential for adaptive immunity as they acquire T-cell receptors (TCRs) in the thymus to recognize foreign antigens from infectious pathogens as well as tumor antigens (Mitchison, 1955; Jorgensen et al., 1992; Park and Renier, 2010). Since 1980s, ex-vivo expanded T-cells have been used for treatment of diseases such as melanoma, cytomegalovirus and HIV (Rosenberg et al., 1988; Riddell et al., 1992; Levine et al., 2002). The initial deployment of T-cells required sorting and expansion of allogeneic or autologous lymphocytes for their reintroduction into patients, yet generation of disease-specific T-cells is cumbersome as patients usually express limited numbers of cells that are reactive against the specific target (Sadelain et al., 2003; Park and Renier, 2010). Using allogeneic T-cells and in some cases autologous Tcells led to high risks of developing graft-vs.-host disease and rejection of infused T-cells. Relying on naturally expressed TCRs requires tumor antigens to be presented by specific major histocompatibility complexes (MHC), which are usually downregulated or dysfunctional in many tumors besides being very specific to each patient (Hicklin et al., 1999; Khong and Restifo, 2002; Park and Renier, 2010).

Engineered T-cells have emerged to better control the safety and effectiveness of T-cell therapies particularly controlling antigen targeting and T-cell function (Sadelain et al., 2003).

#### TABLE 1 | Carrier-free nucleic acid therapeutics in clinical trials.

*(Continued)*

#### TABLE 1 | Continued

*(Continued)*

#### TABLE 1 | Continued

*The data is compiled from www.ClinicalTrials.gov based on nucleic acid keyword search and choosing the trials where no obvious carrier was used.

They represent one of the most advanced therapeutic options as they are a "living drug" which combine major advances in antibody engineering, vaccination and transplantation (Lim and June, 2017). Two T-cell based therapies recently approved by the FDA (National Cancer Institute, 2017), axicabtagene ciloleucel (YescartaTM) 2 and tisagenlecleucel (KymriahTM) 3 , are genetically modified cells to express Chimeric Antigen Receptors (CARs) against CD19, an antigen present throughout the Bcell lineage and one of the first targets for development of monoclonal antibodies (mAb) for B-cell malignancies (Engel et al., 1995; Katz and Herishanu, 2014; Park et al., 2016). Along with targeting "liquid" cancers, they are now being explored to target 'solid' cancers, as well as infectious diseases or undesired immune responses with >250 ongoing clinical trials (Scholler et al., 2012; American Association for Cancer Research, 2017; Maldini et al., 2018).

T-cells have been primarily modified to express CARs by viral gene transfer; in cases of YescartaTM and KymriahTM, replicationdefective gammaretrovirus and lentivirus vectors, respectively, were used for gene transfer (Hu and Pathak, 2000; Sadelain, 2017; Zhang et al., 2017), which enabled permanent transgene insertion into the genome (Hu and Pathak, 2000). However, retroviral gene transfer has been associated with high risk of insertional mutagenesis in the past, especially when vectors get inserted close to growth-control genes, leading to oncogenesis, immune reactions, and other toxicities (Hacein-Bey-Abina et al., 2008; Wang et al., 2008, 2015). The production of viral vectors is also laborious, with production times ranging from 2 weeks to 6 months and differences in batches or sources making it difficult to compare and replicate (Przybylowski et al., 2005; Ivics et al., 2009; Levine et al., 2016; Kebriaei et al., 2017; Zhang et al., 2017). As a result of these drawbacks, non-viral vectors that are easier to synthesize, cheaper, less toxic and more consistent to produce are being constantly developed to match the effectiveness of viral vectors (**Table 2**). SB transposons, just as retroviral vectors, can integrate themselves in the genome and address the issue of longevity of expression (Ivics et al., 2009). The SB transposon system was the first one to be effective in vertebrate cells and since then other transposons that are more active in some cell lines, such as the piggyback transposon, have been investigated (Wu et al., 2006; Muñoz-López and García-Pérez, 2010). Transposons rely on TIRs that are recognized by transposases to "cut" and "paste" the gene at desired destinations. Thus, a transposon vector with the gene of interest with the TIRs and a transposase-coding pDNA or mRNA need to be delivered to target cells (Yant et al., 2000; Wu et al., 2006). While transposon systems could be simpler and more predictable with lower risk of immunogenicity (Walisko et al., 2008), transposable elements are not free of risks of genotoxicity and they still rely on carriers for transport through the cell membrane. Viral vectors are still being used for transposons with the same challenges discussed before (Doudna and Charpentier, 2014; Boehme et al., 2016; Richter et al., 2016). Other gene editing technologies include designer nucleases, including zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR/Cas9 system which induce double strand breaks in a target site followed by the addition of a gene of interest (Urnov et al., 2010; Gaj et al., 2013; Jung and Lee, 2018). The CRISPR/Cas9 system is already being tested in clinical trials in China and CAR T-cell engineering in the US (Svoboda et al., 2018). The designer nucleases depend on cellular enzymes for gene insertion that require dividing cells in contrast to some integrating viruses and transposon systems that can also target non-dividing cells (Di Stasi et al., 2011).

2YescartaTM (axicabtagene ciloleucel) suspension for intravenous infusion Initial U.S. Approval: 2017. Available online at: https://www.fda.gov/files/vaccines%2C %20blood%20%26%20biologics/published/Package-Insert---YESCARTA.pdf 3KymriahTM (tisagenlecleucel), first-in-class CAR-T therapy from Novartis, receives second FDA approval to treat appropriate r/r patients with large B-cell lymphoma (2018). Available online at: https://www.novartis.com/news/media-releases/ kymriahr-tisagenlecleucel-first-class-car-t-therapy-from-novartis-receivessecond-fda-approval-treat-appropriate-rr-patients-large-b-cell-lymphoma

TABLE 2 | Broad comparison of viral and non-viral transfection vectors.

One of the main challenges associated with shifting to nonviral methods for transfection of T-cells is the inability of nonviral systems to match the efficiency of viral systems, especially in terms of longevity of gene expression. However, the transient expression by non-viral vectors may be advantageous as it may lead to reduced side effects and complications for patients (Hardee et al., 2017). Another challenge for non-viral vectors in T-cell engineering is less than optimal in vivo targeting and continuous stimulation that must be provided by the material to the cells, which is inherently difficult to achieve as these cells are present in suspension, are constantly dividing and usually exist in an immunosuppressive environment (Zheng et al., 2013; Ansari et al., 2017). Thus, innovative biomaterials that find solutions to these two challenges are of paramount importance in the field and can also serve as a point of reference for biomaterials targeting various cells besides T-cells. The alternative to viral modification of T-cells is commonly based on membrane pore-inducing electroporation/nucleofection without any carriers. Ramanayake et al. recently compared the average costs of viral delivery (\$3- 500,000) with an electroporation/transposon approach (\$6,000) to produce CAR T-cells under GMP conditions. By optimizing electroporation conditions, modified CAR T-cells persisted in the peripheral blood for >3 weeks and transgene expression was >50% (Wells, 2004). A safety guard included in their transposon sequence was the inducible caspase 9 suicide gene, which directs targeted elimination of engineered T-cells by administration of a small molecule (Wang et al., 2017). Some drawbacks of electroporation, however, are toxicity and difficulty for in vivo applications due to limited access to target sites (Holstein et al., 2018). Longer ex vivo expansion might be required to allow cells to recover from undesirable consequences of electroporation, since grafting nucleofected hematopoietic cells in a preclinical model was improved with longer culture times (Holstein et al., 2018). An alternative approach to electroporation is "cell squeezing" using microfluidic devices that rely on rapid mechanical deformation of cells to passively introduce genes and materials of interest (Sharei et al., 2013a,b).

In a similar manner, as more precise control is desired for immunomodulation of T-cells, diverse biomaterials and nanotechnologies have emerged as platforms to address major stages and challenges of CAR T-cell development, namely ex vivo and in vivo expansion of cells and CAR gene delivery (**Table 2**). The majority of T-cell therapies involve ex vivo expansion of modified/to be modified cells but not all expanded T-cells have the same therapeutic efficacy (Fraietta et al., 2018). T-cell expansion most commonly employs commercially available polystyrene microbeads (Dynabeads) that aim to simulate the action of antigen presenting cells (APCs) targeting T-cell activation through CD3 and CD28 stimulus and IL-2 supplementation (Kalamasz et al., 2004; Hollyman et al., 2009; Li and Kurlander, 2010). However, these non-degradable beads need to be separated prior to cell delivery to patients and they also dysregulate some T-cell functions as their mode of action is not as close as APC activation. Other alternatives to naturally derived APCs include poly(lactide-coglycolide) (PLGA) microparticles (Steenblock and Fahmy, 2008), phosphatidylcholine and cholesterol liposomal systems (Prakken et al., 2000), paramagnetic iron-dextran NPs (Perica et al., 2014), polydimethylsiloxane (PDMS) microbeads (Lambert et al., 2017) and carbon nanotubes composites with PLGA NPs (Fadel et al., 2014), highlighting the compatibility of various systems with T-cell stimulation and also how different physiochemical properties of the particles may have different efficiencies on Tcell expansion. Besides the use of NP systems, 3D scaffolds have also been developed which can be used as implants at tumor sites.  With such broad possibilities, the versatility of biomaterials to design biomimetic systems to effectively expand T-cells is evident. Scaffolds have also been used as in vivo immunomodulation niches promoting sustained release and expansion of T-cells directly at tumor sites. For example, an alginate scaffold was reported that delivered T-cells and a STING (Stimulator of Interferon Genes) agonist, serving as a vaccine in pancreatic and melanoma mice models (Smith et al., 2017a). Co-delivery of T-cells and STING agonist not only eradicated the tumors in some mice, but also enabled the cured mice to develop a systemic antitumor immune response and resistance to metastasis when re-challenged with pancreatic tumor cells (Kim et al., 2014). One of the limitations of implantable scaffolds is that they require surgery to be introduced to desired sites, but to address this, injectable formulations that form scaffolds in situ are being developed. Injectable mesoporous silica rods were able to spontaneously form scaffolds, recruit APCs and subsequently elicit specific T-cell responses (Kim et al., 2014).

To deliver the CAR genes, synthetic carriers have also been developed (**Table 3**) with the aim of reducing viralinduced reactions, while increasing delivery loads and ease of manufacture (Zhou et al., 2017). Synthetic carriers may need to be modified with targeting antibodies, peptides or recombinant molecules that augment their transfection specificity in hard-totransfect cells growing in suspension (Liu et al., 2018). So far, lipid (Moon et al., 2011) and polymeric (Smith et al., 2017b; Olden et al., 2018) delivery systems have been used for generating CAR T-cells with targeting capacity inducing tumor regression in a mouse model (Smith et al., 2017b). To our knowledge only two groups have reported in vivo generation of CAR Tcells, the Buchholz group at the Paul-Ehrlich-Institut and the Stephan group at the Fred Hutchinson Cancer Research Center. The Buchholz group reported a lentiviral approach targeting CD8 receptors and the Stephan group utilized poly(β-amino ester) NPs, both successfully generating CD19-CAR T cells in vivo (Smith et al., 2017b; Pfeiffer et al., 2018). Comparing the two approaches, the lentiviral approach allowed for greater percentage of CAR T-cell generation of up to 35% in blood and the synthetic NPs reported up to ∼20% transfection. In addition, both studies compared their in vivo targeting systems to infusions with conventionally generated CAR T-cells ex-vivo and did not find significant differences between the two treatments. This opens up a new avenue to increase the efficiency of CAR T-cell engineering and avoid the cumbersome ex vivo expansion and reprogramming steps. It is to be noted, however, that even though both approaches established significant advances in the efficiency of CAR T-cell generation, they still encountered some of the main challenges of the therapy including Bcell depletion and signs of cytokine release syndrome with the use of lentivirus. B-cell depletion arises since CD-19 is not only present in leukemic cells but also in non-leukemic B-cells, highlighting the hurdle of finding the right antigen to target.

As more advances are reported for CAR T-cell technologies, some common challenges have emerged. The treatment of solid tumors is one such major challenge, as the tumor microenvironment is highly immunosuppressive due to combination of down-regulated tumor antigens and T-cell suppression (Joyce and Fearon, 2015; Cheung et al., 2018). However, the in situ scaffold approaches aim to reverse the immunosuppressive environment by sustained release of cytokines for recruitment of immune cells at tumor sites. Once the cells are transferred to patients, T-cells act autonomously and so far it is very difficult to control their actions and unwanted side effects in-situ [e.g., cytokine release syndrome, neurotoxicity or B-cell aplasia (Yant et al., 2000)], so that feedback systems are needed to better control the therapy (Lim and June, 2017). As CAR T-cell technologies continue to progress, an interdisciplinary effort must be made to address some of the pressing challenges that include their mode of delivery and expansion, migration, and mechanism of action so as to exert their action on tumor cells while sparing the normal cells.

# BIOMATERIALS AND EMERGING NUCLEIC ACID TECHNOLOGIES

Biomaterials have been an integral part of emerging cell and gene based technologies over the years. Early work on skin substitutes, for example, laid the foundation for the tissue engineering field by relying on biomaterials to create the right milieu to allow tissue-like organization of seeded (ex vivo) or invading (in vivo) cells (Bell et al., 1979; Yannas, 1992), while separate efforts were being undertaken to devise ingenious ways to transfer foreign genes into tissues by using projectiles to penetrate the skin (Williams et al., 1991). The amalgamation of separate approaches allowed biomaterials to support tissue organization ex vivo and to implement new gene transfer techniques, resulting in ex vivo construction of devices from gene-modified cells for transplantation (Tai and Sun, 1993). From these beginnings, biomaterials have evolved to now enable several key technologies at the center of nucleic acid-based therapies. Below we summarize the impact of biomaterials in key areas important for the future of nucleic acid therapeutics.

# More Intelligent NPs (Figure 2)

Increasing complexity (i.e., functionalization) in nucleic acid bearing NPs will be the way forward to realize more effective therapeutic outcomes from nucleic acids. Despite emergence of a wide range of synthetic, "intelligent" materials for NP fabrication in the last decade, there is still a need to create new functional NPs for hard-to-transfect cell types. Using commercial and in-house developed non-viral reagents, the authors frequently encounter cell types (e.g., leukemic stem cell lines and certain mesenchymal stem cells) that are exceptionally difficult to transfect. Patientderived cells in particular have shown variable results in our hands for siRNA-mediated silencing of therapeutic targets, with significant fraction of cells either not responding or responding weakly to nucleic acid treatments (unpublished observations, and Gul-Uludag et al., 2014; Landry et al., 2016; Valencia-Serna et al., 2019). We recognize that increasing complexity in NP design, while improving performance, places extra burden on manufacturing processes, so that new design features amenable for scale-up will be especially critical for clinical translation.

Packaging nucleic acids with a combination of cationic and lipidic biomaterials have been recognized to improve delivery as compared to either moiety alone (Incani et al., 2010; Liu et al., 2010). Additional functionalization of NPs has been possible with antibodies (Kedmi et al., 2018) and other ligands (Guan et al., 2019) using lipidic anchors, and peptides/proteins by electrostatic anchors on self-assembled systems (Dong et al., 2018). Excessive cationic charge density, a recognized limitation of NPs, could be altered by incorporating anionic macromolecules into NPs. We incorporated anionic hyaluronic acid into NPs, either as a surface coating or additive into the core, that controlled the -potential of NPs in a predictable way as well as increased the propensity of NPs for dissociation that was beneficial for both pDNA (Remant Bahadur et al., 2015) and siRNA delivery (Parmar et al., 2018). Another benefit was improved stability of NPs (Rose et al., 2013), with direct implications for in vivo administration. This benefit was not unique for multivalent polymeric carriers, but even liposomal systems such as the commercially available FugeneTM which derived a beneficial effect from the additives in complexes (Nakamura et al., 2015); the additives in this case were hydrophilic/uncharged PEG and anionic tRNA that were widely different in molecular features, yet they were both able to enhance the transcriptional activity of a minimal PCRamplified DNA expression cassette in the robust HEK293 cells (Nakamura et al., 2015). Different mechanisms might therefore be responsible (or effective) to weaken nucleic acid binding just enough to enhance the availability of nucleic acids intracellularly. Alternatively, the cationic charge density initially required for nucleic acid complexation could be removed by a controlled

chemical cleavage (Jiang et al., 2019), while the NPs are retained in place by covalent linkages or possibly by other affinity interactions such as the hydrophobic domains. Improved toxicity was reported against a well-recognized liposomal formulation as a result of charge reduction, but systematic studies on the beneficial effect of reducing cationic charge density remains to be reported (Jiang et al., 2019).

Self-assembly has been favored in the hands of most researchers due to its convenience to create NPs at the time and site of application, in addition to the possibility of seamlessly incorporating additional functional molecules into the NPs. However, pre-manufactured NPs that bear nucleic acids may reduce variability associated with "on-the-spot" NP preparations and improve stability during the delivery. Nanogels, physically or chemically crosslinked polymeric networks with high water content, are emerging as leading candidates in this regard (Zilkowski et al., 2019). Nanogels with targeting ligands can entrap nucleic acids by electrostatic interactions or "irreversible" covalent linkages. Cargo can be loaded during synthesis or post-synthesis. Compared to conventional hydrolyticallydegrading NPs, nanogels offer the possibility of more robust degradation under defined redox, pH and microenvironmental conditions, leaving behind a smaller footprint. To create a biomimetic means to shield the excess cationic charge of nanogels, they have been decorated with "recognizable" polysaccharide chains in a way replacing the synthetic PEG decoration. Polysaccharide chains can undergo degradation at sites of specific enzymatic activity (Nishimura et al., 2017), so that cellular uptake is facilitated at these sites, preventing non-specific interactions caused by the cationic charge in other (especially serum) sites. Recently, NPs prepared with adenosine-5 ′ -triphosphate (ATP) responsive phenylboronic acid (PBA) bearing polymers (Naito et al., 2012) or ATP-responsive aptamers (Mo et al., 2015) are providing new ways of releasing the cargo intracellularly in response to high cellular ATP concentration that is typically absent in the extracellular space. The ATP-triggered release is reminiscent of the glutathione (GSH)-sensitive disulfide linkages, an earlier approach for intracellular cargo release

triggered by the severe GSH gradient between the intracellular and extracellular compartments. The latter approach seems simpler to implement but the relative efficiency of intracellular vs. extracellular cleavage rates under physiological conditions for the two approaches remains to be thoroughly compared. Both of these approaches rely on physiological stimuli to execute the cargo release. If one wishes to rely on an external trigger for cargo release, analogous to inducer-activated gene expression or silencing, Khan et al. have recently reported an externally activated approach to nucleic acid release (Khan et al., 2017), whereby the small molecule tetrazine was capable of breaking the trans-cyclooctene linkages holding onto siRNAs in a NP. The relative stability of the trans-cyclooctene linkage and its specificity to tetrazine cleavage was proposed as a superior 'ondemand' release of nucleic acids, where the proof-of-principle studies were reported in cell culture conditions.

Finally, another approach to intelligent NPs proposed by Mirkin group is to create spherical nucleic acids (SNAs) assembled on NP cores; they were shown to effectively penetrate the blood-brain-barrier as well as the blood-tumor-barrier and implement the RNAi silencing pathway (Cutler et al., 2012; Young et al., 2012; Jensen et al., 2013; Li et al., 2018). This seems to be possible with clustering of polynucleotides (which by themselves do not effectively cross cell membrane), perhaps due to an increased fluid phase uptake of the NP configuration and/or the lipophilic NP core.

# mRNA Delivery to Replace pDNA Therapy

mRNA delivery has been pursued for some time now with successful mRNA transfer by lipidic carriers reported as early as 1989 (Malone et al., 1989). Recent efforts to modify the nucleic acid for improved stability, better translation and lower immunogenicity are opening up new possibilities for its broader deployment (Kormann et al., 2011). Given the wealth of already developed carriers for other types of nucleic acids, a critical issue is whether we need new carriers for mRNA delivery or are the previous carriers sufficient to deploy this particular nucleic acid. While debatable, new carriers that rely on charge alteration to reduce/eliminate the electrostatic binding to mRNA and making mRNA freely available to translation machinery have been reported (McKinlay et al., 2017). Even with these apparently effective carriers, the outcome from in vivo mRNA administration is short-lived, with expression levels returning to baseline levels within ∼48 h time frame. Some studies indicate that carriers previously developed for other nucleic acids can be employed, and in head-to-head comparisons, optimized pDNA delivery could be even superior to mRNA delivery in some cases, for example with human bronchial epithelial cells and lung delivery in vivo (Guan et al., 2019). Other studies reported the opposite; when comparing mRNA vs. pDNA delivery, biomaterial scaffolds were reported to display superior mRNAinduced transgene expression for a longer duration in vitro (Elangovan et al., 2015; Balmayor et al., 2016). The nature of the delivered gene and its regulation, the nature of the carrier (i.e., its influence on intracellular pharmacokinetics of the cargo) as well as the specific cellular system (i.e., in particular endocytosis efficiency against different cargos and ability for nuclear import) could be the reason(s) for the observed differences. It is likely that minicircle pDNA (that bear no non-essential genetic elements) with improved design over the traditional pDNA could be superior over the mRNA based gene expression, while mRNA could be superior over the traditional pDNAs. On the other hand, optimization of terminal repeats and/or incorporated modified bases make significant differences in mRNA performance, so that the effectiveness of mRNA over conventional pDNAs may be variable in different systems and this may take some time to clarify. Our own experience indicates that relative performance of pDNA vs. mRNA is cell-line dependent, and that some cells display better transgene expression from mRNA polyplexes, while others provide more robust expression from pDNA (unpublished). This observation possibly reflects the nuclear import capability of the cells, their proliferation rate and/or the capability of the carrier to deliver the pDNA to the nucleus.

Vaccination seems to be an especially appropriate area for mRNA administration where the adjuvant ability of mRNA may be additionally beneficial for a strong transient response. Implants where the biomaterials act as a local matrix (scaffold) to modulate the release of mRNA are an effective approach to vaccination (Chen et al., 2018), especially if prolonged local presence and/or controlled release is optimal. Scaffolds could be viewed as passive carriers of mRNA particles; transfection reagents are usually designed to transfect cells with no specific consideration to scaffolds (Steinle et al., 2018). The avidity (i.e., overall strength) between the nucleic acid and the complexing biomaterial has been shown to control mRNA release from NP formulations (Lallana et al., 2017) and it is likely that such a relationship will hold true for macroscopic scaffolds as well. In a presumably continuous scaffold, this will require control over the density of charges if no other "binders" are considered. The relatively weak immuneadjuvant features of mRNA could be further improved by employing double stranded mRNAs that are highly recognized by pattern recognition receptors (PRRs); in this case, an optimal length of double strands was needed to balance the immunostimulation with translational activity (Uchida et al., 2018). Alternatively, polymer-condensed mRNA could be entrapped in lipidic envelopes to enhance uptake and adjuvant activity (Persano et al., 2017).

Bone induction by mRNA translation is another indication where transient transgene expression might be sufficient for clinical success. Morphogens such as Bone Morphogenetic Proteins (BMPs) are known to "kick-start" the osteogenesis process beyond a critical concentration and their continued presence might not be required to sustain tissue induction and repair. The precise design of mRNA with particular chemical and end-group modifications are critical for effective translation, but several successful configurations have emerged for relatively long term protein production indicating some flexibility in the mRNA design. A longer sustained expression was noted when a BMP-2 morphogen was delivered with mRNA in scaffolds, presumably reflecting favorable pharmacokinetics and cell exposure (i.e., gradual vs. bolus) (Balmayor et al., 2017; Guan et al., 2019). Recent studies led by Balmayor et al. (2017), Badieyan et al. (2016) and Zhang et al. (2019) employed small animal models to assess the potential of mRNA-based bone repair, with so called "transcript activated matrices" (TAMs). A range of cells including easy-to-transfect cell lines and primary cells derived from adipose tissue and bone marrow were effectively induced for mRNA translation and significant secretion of therapeutic proteins (Badieyan et al., 2016; Balmayor et al., 2017; Zhang et al., 2019). It appears that robust effects were obtained even though the scaffolds were not optimized for bone repair (Elangovan et al., 2015). The elimination of the additional nuclear import barrier in primary cells, which is the limiting step for pDNA delivery, is an important advantage for deploying mRNA and makes this nucleic acid the preferred agent for delivery. Older studies, however, also showed some bone repair with pDNA based systems (so called "gene activated matrices," GAMs) in similar preclinical models (Ono et al., 2004; Huang et al., 2005; Zhang et al., 2009; Qiao et al., 2013). Limited regeneration was noted in early investigations with BMP-4 pDNA/PEI25 (25 kDa branched PEI) implants around defect edges (Huang et al., 2005) potentially due to toxicities of high pDNA/PEI25 dose (200 µg of pDNA and likely >200 µg of PEI25) (Plonka et al., 2017; Khorsand et al., 2019). Ono et al. employed a hydroxyapatite scaffold to deliver cationic liposome condensed pDNA in a rabbit cranial defect, where the BMP-2 pDNA induced new bone tissue had penetrated halfway into the defect after 9 weeks (Ono et al., 2004). Qiao et al. employed PLGA particles containing BMP-2 pDNA/PEI25 and gelatin sponges in a calvarial defect model; bone formation was stimulated by BMP-2 gene delivery at defect edges (Qiao et al., 2013). More recently, in a head-to-head comparison, a GAM with pDNA and a TAM with mRNA for BMP-9 expression were found to be equivalent for bone induction in vivo (Khorsand et al., 2019), again suggesting no clear impediment to pDNA based GAMs in tissue induction. While difficult to compare these independent studies, the authors believe that mRNA may provide more robust osteogenic transformation in vivo (given the lower dose of mRNA in implants vs. pDNA), leading to comparatively better results in certain animal models. Lower doses of nucleic acids/synthetic carriers may minimize adverse inflammatory/immune reactions that may impede new tissue induction. The delivery system used in mRNA delivery were not particularly tailored in these early studies (i.e., PEI and a cationic lipid), so that improved carriers are bound to further improve regeneration with reduced doses. Collectively, these studies indicate that a robust translation of mRNA for ∼10 days in vitro appears to be sufficient for effective tissue induction in small animal models. Investigations in larger animals, however, will be required to truly assess clinical potential. Considering that µg quantities of BMP proteins are needed for effective regeneration in small animals and that clinical therapy in the past relied on 10–20 mg of the protein in situ, it will be important to determine the functional mRNA doses in larger preclinical models to better assess its potential for clinical translation.

# Long-Acting Gene Expression With Non-viral Systems

The emerging T-cell therapy has again shined a light on the need for long-term gene expression with non-viral approaches. Transposons have emerged as a viable alternative to integrating viruses to this end whose utility is now being tested in clinical studies (Kebriaei et al., 2016; Tipanee et al., 2017b). The SB system relies on integration-enabling transposases, which can be delivered in protein form, in an expression plasmid, including the minicircle approach (Holstein et al., 2018) or more recently with mRNA (Monjezi et al., 2017). The latter approach obviates the need for nuclear delivery and may be a superior alternative due to transient induction of a transposase that will limit longterm transposition and hence unpredictable events. The current process of transposon delivery operates with nucleofection, which is a special form of electroporation with "facilitating" buffers. Although effective, nucleofection process is associated with loss of viability in a significant proportion of treated cells, so that it hampers ex vivo expansion efforts and prolongs attainment of critical mass of cells needed for transplantation. With an optimized combination of expression/integration system, 25– 35% of cells were shown to retain the transgene expression in hard to transfect CD34+ hematopoietic stem and progenitor cells (HSPCs) (Holstein et al., 2018), based on the assessment of transgene expression in HSPC colonies or vector copy integrated/diploid genome. From a safety perspective, integration profiles of the transgene were favorable for the SB system over lentiviral vectors in human HSPCs, leading to random integration away from transcriptional regulatory elements of active genes and other "hotspots." However, with integrating vectors, a finite risk of long-term adverse effects is present and should be considered in the face of benefit to be derived (Moffett et al., 2018).

Implementing transposon-based long-term gene expression in vivo will be desirable but also particularly challenging. Toward this goal, a NP system was described that were deigned to transfect T-cells (functionalized with an anti-CD3e f(ab')2 fragment) in a murine model (Smith et al., 2017b); in vitro results indicated a relatively low level of transfection (∼4% of population), but this was sufficient for target cell killing and matched the performance of lentiviral-modified T-cells. The low levels of transfection will translate into benefit in terms of lower non-specific binding (and modification) of non-target cells. The extent of in vivo modification was similar with ∼5% of circulating T-cells displaying transgene expression after 6 days, but the cells expanded with increasing population of cells displaying CAR expression (∼20% after day 12), that was dependent on transposase delivery. In the absence of transposase delivery, no effective anti-tumor response was seen, clearly indicating the beneficial effect of vector integration. A similar delivery system was used for transiently transfecting T-cells with mRNA, whose biocompatibility was compared to electroporation modified cells (Weiden et al., 2018a); the modification with the non-viral system was implemented with lower adverse effects on cells, as evident in subsequent expansion rate ex vivo. It was interesting to note that this study also used a transiently expressed transcription factor Foxo1 (from mRNA whose expression lasted for ∼5 days) that favors the expansion of desirable population of T-cells, that may provide a superior alternative to transient delivery systems towards the ultimate goal of integrated (long-acting) vectors (Broderick and Humeau, 2017).

# Expanding Genetically Modified Cells

Irrespective of the modification approach, CAR and other genetically modified cells may need to be expanded to provide them with a survival advantage when grafted into a host. This has been implemented in the past by using soluble cytokines, intracellular expressed factors (Weiden et al., 2018a) and immobilized ligands on tissue culture surfaces. The "Dynabead" system with immobilized CD3/CD28 antibodies on microparticles has been commercialized towards this end. Alternatively, one can employ biomaterials scaffold-conjugated ligands to enhance stimulation over that of soluble cytokines, and avoid additional manipulation of cells for transcription factor

expression. Hydrogels derivatized with α2β1 collagen receptor binding GFOGER peptide or multiple integrin-binding RGD motif have been described that support T-cell expansion in vitro and housing after grafting the cells in vivo (Cheung et al., 2018; Weiden et al., 2018b). The hydrogels could immobilize ligands or provide local release of cytokines important for cell expansion (**Figure 3**), which may be difficult to implement with systemically administered agents. Infiltrating cells can be stimulated and expanded within designer niches (Ren and Lim, 2018). Delivering anti-CTLA-4 and anti-OX40 mAbs has been described to stimulate tumor-infiltrating killer T-cells with scaffolds in the vicinity of resected tumors (Wang et al., 2016). It has been possible to create scaffolds from nucleic acids (DNAbased) to release immune stimulatory PD-1 blocking agents (Lynn et al., 2019). The importance of size, architecture and ligand density, among others, are beginning to be elucidated for in vivo expansion of T-cells (Liu et al., 2018), while a similar approach is implemented for ex vivo expansion. It has been recognized for some time that immobilized antibodies are more potent in stimulating T-cell expansion compared to soluble ligands, and a mechanosensor receptor (Piezo1) was recently identified as a mediator of TCR activation (Zhang et al., 2018). This provides a mechanistic link on how mechanical properties of a scaffold could affect T-cell stimulation and expansion directly, and may provide a more rational design of the biomaterials scaffold to optimize TCR activation and T-cell expansion.

Vaccination with pDNA is continuing to be explored with biomaterial-based delivery and adjuvant systems (Zhang et al., 2018), with muscle and skin sites (by electroporation) popularly used for in situ expression of tumor antigens from pDNA directly (Amante et al., 2015; Broderick and Humeau, 2017). Inhibition of immune checkpoints CTLA-4 and PD-1 is making inroads to enhance the anti-tumor response with in situ expressed tumor antigens (Lopes et al., 2018). Simple injection of pDNA without the use of electroporation has been made effective with the use of a combination of cationic lipid formulations, where the 2-dis-tearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(PEG-2000)] was critical in supporting expression of antigens and long-term antibody response (Ho et al., 2018). Hydrolytically-cleaving polyesters have also been shown to successfully elicit effective antibody response against pDNAcoded antigens, with a lipid-modifed PEI (PEI1.8-deoxycholic acid) facilitating local transfection and antigen expression (Giang Phan et al., 2019).

# CONCLUDING REMARKS ON FUTURE NUCLEIC ACID THERAPIES

Synthetic, precisely engineered biomaterials and self-assembled systems from such biomaterials are leading the way to enable a

# REFERENCES

diverse array of therapeutic modalities that rely on nucleic acids. The prospect of improved clinical safety of the biomaterial-based delivery is driving this endeavor and significant efforts are in place now to enhance the effectiveness of the delivery, while allowing a high degree of modification of "hard-to-transfect" cells and realizing permanent modification (whether it may be transgene expression or silencing). Nucleic acids themselves derived from DNA and RNA molecules have the potential to replace synthetic biomaterials and act as carriers for nucleic acid agents (Hu et al., 2018). It has been possible to create responsive systems to release different effector molecules from a scaffold of nucleic acids with precise controlled features. One can envision delivering CpG oligodeoxynucleotides, that bind TLR9, from DNA scaffolds effortlessly to stimulate dendritic cells against tumors (Bourquin et al., 2008). The possibilities are diverse, but whether they can be produced in economical terms, their in vivo stability be controlled and adverse reactions in situ be minimized remains to be seen for such nucleic acid scaffolds. While delivery with biomaterials for therapeutic purposes has been the main focus, one can envision relying on nucleic acids for "preventative" medicine as well; with the identification of aberrant genes and/or miRs before manifestation of clinical symptoms, one has the opportunity to employ nucleic acids before disease development. One can envision deleting "aberrant" cells or restoring normal physiology ahead of detectable symptoms. Perhaps our next generation of "vitamins" will be based on nucleic acids as preventative remedies; nevertheless, the functional use of nucleic acids will rely on designer biomaterials and nano-engineered systems in order to present the nucleic acids to the appropriate cells in the appropriate manner.

# AUTHOR CONTRIBUTIONS

All authors contributed to the conceptualization, literature search/review, and writing of the article. Final editing was undertaken by HU.

# FUNDING

The gene therapy and nucleic acid studies in the authors lab have been supported by operating grants from Canadian Institutes of Health Research (CIHR), Canadian Breast Cancer Foundation (CBCF), Natural Sciences and Engineering Council of Canada (NSERC), Women and Children Health Research Institute (University of Alberta), Alberta Innovates (AI), and the University of Alberta Office of Research. Past infrastructure support was provided by AI, Edmonton Civic Employees and Canadian Foundation for Innovation (CFI). We thank numerous current and past trainees, and collaborators for contributing to the evolution of our ideas on this review topic.

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**Conflict of Interest Statement:** HU is a founder and share holder in a private company (RJH Biosciences Inc.) intended to develop nucleic acid based therapies and declares conflict of interest.

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 Uludag, Ubeda and Ansari. 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.

# Corneal Repair and Regeneration: Current Concepts and Future Directions

Mohammadmahdi Mobaraki 1 , Reza Abbasi 1 , Sajjad Omidian Vandchali 1 , Maryam Ghaffari 1 , Fathollah Moztarzadeh1 and Masoud Mozafari 2 \*

1 Biomaterials Group, Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran, 2 Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

The cornea is a unique tissue and the most powerful focusing element of the eye, known as a window to the eye. Infectious or non-infectious diseases might cause severe visual impairments that need medical intervention to restore patients' vision. The most prominent characteristics of the cornea are its mechanical strength and transparency, which are indeed the most important criteria considerations when reconstructing the injured cornea. Corneal strength comes from about 200 collagen lamellae which criss-cross the cornea in different directions and comprise nearly 90% of the thickness of the cornea. Regarding corneal transparency, the specific characteristics of the cornea include its immune and angiogenic privilege besides its limbus zone. On the other hand, angiogenic privilege involves several active cascades in which anti-angiogenic factors are produced to compensate for the enhanced production of proangiogenic factors after wound healing. Limbus of the cornea forms a border between the corneal and conjunctival epithelium, and its limbal stem cells (LSCs) are essential in maintenance and repair of the adult cornea through its support of corneal epithelial tissue repair and regeneration. As a result, the main factors which threaten the corneal clarity are inflammatory reactions, neovascularization, and limbal deficiency. In fact, the influx of inflammatory cells causes scar formation and destruction of the limbus zone. Current studies about wound healing treatment focus on corneal characteristics such as the immune response, angiogenesis, and cell signaling. In this review, studied topics related to wound healing and new approaches in cornea regeneration, which are mostly related to the criteria mentioned above, will be discussed.

Keywords: cornea, tissue engineering, wound healing, regenerative medicine, biomaterials, immune privilege, angiogenesis, limbus

# INTRODUCTION

Diseases affecting the cornea can be either infectious or non-infectious, and both may cause severe visual impairments requiring intervention. Trachoma, onchocerciasis, corneal ulceration, corneal dystrophies, and xerophthalmia are some of the major causes of blindness worldwide (Sommer, 1982). However, the prevalence and epidemiology of corneal diseases varies from region to region.

#### Edited by:

Hasan Uludag, University of Alberta, Canada

#### Reviewed by:

Julien Georges Didier Barthès, Protip Medical, France Emilio Isaac Alarcon, University of Ottawa, Canada

\*Correspondence: Masoud Mozafari<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 14 December 2018 Accepted: 20 May 2019 Published: 11 June 2019

#### Citation:

Mobaraki M, Abbasi R, Omidian Vandchali S, Ghaffari M, Moztarzadeh F and Mozafari M (2019) Corneal Repair and Regeneration: Current Concepts and Future Directions. Front. Bioeng. Biotechnol. 7:135. doi: 10.3389/fbioe.2019.00135 From the use of traditional eye medicines (which is now considered a significant risk factor for corneal ulceration) to collagen cross-linking, which has recently been approved by the US FDA to strengthen the cornea, the ultimate goal in corneal treatment is to employ minimally invasive procedures that can restore or preserve vision (Jeng et al., 2016). Stimulating the body's repair mechanisms is now considered to be the gold standard for the functional healing of damaged tissues and organs (Khadem et al., 2000, 2004; Zarrintaj et al., 2017). This approach is typified by corneal transplantation and tissue engineering.

The most striking advance in the medical treatment of corneal diseases over recent decades has been corneal transplantation and, nowadays, the cornea is the most commonly transplanted tissue worldwide. Corneal transplantations are divided into two main categories based upon the amount of surgically replaced tissue. In penetrating keratoplasty, the entire cornea is replaced with a donor tissue. However, in a newer procedure called lamellar keratoplasty, only the damaged layers are replaced with a donor graft, and the healthy part of the cornea is left intact. In lamellar keratoplasty, the integrity of the cornea and the surrounding tissues are preserved; as a result, better visual improvement is usually achieved. Unfortunately, there are sometimes poor outcomes because of graft rejection or late graft failure. Furthermore, according to a World Health Organization (WHO) report, 15–20% of patients who need corneal transplantation remain untreated because of the shortage of corneal donors (Whitcher et al., 2001). Over the last few decades, the shortage of donor tissues, beside fears of transmissible diseases, has accelerated studies on finding an alternative treatment to transplantation, and an artificial cornea or keratoprosthesis has been suggested as an option. Historically, Guillaume Pellier de Quengsy Jr. was the first person who proposed a thin silver-rimmed convex glass disc as an artificial cornea, as long ago as 1789 (Mannis and Mannis, 1999). At that time, the first priority was to choose transparent and non-irritating materials, but as time went by other researchers concentrated on designing an artificial cornea which was able to promote better incorporation with the host tissue. Glass and quartz were the choices for the transparent part of the prosthesis and natural polymers like gutta-percha and casein were added to the artificial cornea design. Later, gold rings and platinum rings were used to achieve better incorporation with the host cornea. Further studies on artificial corneas resulted in replacing glass and quartz with lighter materials. At the beginning of the twentieth century, attention was diverted from artificial corneas to transplantation of donor corneal after the first successful keratoplasty (Zirm, 1989). Nevertheless, studies on artificial corneas have never ceased. Today there are four types of keratoprostheses, which are in commercial use. In the following section, these keratoprostheses will be further discussed. Despite some clinical success in using artificial corneas, the host rejection is still relatively high. The presence of corneal epithelial stem cells, which are located in the basal epithelial layer of the corneal limbus (the border between the cornea and the sclera), has given some hope for better healing and integration. Therefore, many researchers have focused on employing new biomaterials to mimic the corneal architecture, which would allow better corneal self-repair. This review will discuss recent advances in repairing damaged corneas, focusing on new concepts and biomaterials (**Figure 1**; Muijzer et al., 2019).

# CORNEA STRUCTURE AND TRANSPARENCY

The human cornea is a unique tissue with two critical functions. On the one hand, the cornea forms the anterior portion of the outer casing of the eye and protects the inner portion of the eye from the external environment. On the other hand, it is the single most powerful focusing element of the eye. It provides about 80% refractive power of the eye and it is roughly twice as powerful as the lens. Because of these functions, the cornea is both mechanically strong and transparent. Its strength comes from about 200 collagen lamellae, which crisscross the cornea in different directions. This collagen-rich layer of the cornea comprises nearly 90% of the thickness of the cornea and is called the "stroma proper layer." In fact, the human cornea is composed of five primary layers—epithelium, Bowman's layer, stroma proper, Descemet's membrane, and endothelium. This hierarchical structure can be described as a fibril-reinforced laminate biocomposite, which provides an excellent compromise between stiffness, strength, toughness, and extensibility. In **Figure 2** the hierarchical structure of the cornea, with macroscopic, microscopic, and nanoscopic features, is shown (Kaufman et al., 2011). As noted earlier, the cornea is transparent; however, the hierarchical structure and the presence of interfaces between the different layers, each with its own index of refraction, seem to be in contradiction with its clarity. There have been lots of studies attempting to explain the transparency of the cornea. The first explanations in the nineteenth century emphasized the homogeneity of the cornea and the fact that the collagen fibrils had the same refractive index. After about a century, Dr. Maurice (1957) tried to explain the optical structure of the cornea considering its geometric form, dimensions and the refractive indices of its components. He suggested that the scattered waves from collagen fibrils interfered with each other in such a way that they canceled out each other in all directions, except the forward direction. Meanwhile, Miller and Benedek (1973) in 1973 showed that the gaps between the interfaces were smaller than one-half the wavelength of visible light; and as a result, the cornea is crystal clear. Recently Meek and Knupp (2015) have reviewed the current state of knowledge about the corneal architecture and its optical transparency. These authors outlined the general basis and molecular mechanisms of corneal transparency. Maurice's suggestion regarding the interaction of the incoming electromagnetic waves with the collagen fibrils was proved to be correct by new imaging technologies (Quantock et al., 2015).

# CORNEAL REPLACEMENTS

Boston Keratoprosthesis (B-KPro), Osteo-Odonto-Keratoprosthesis (OOKP), AlphaCor, and the KeraKlear Artificial Cornea are the four types of keratoprostheses which

have, so far, been commercialized. Over the last decade, implantation of artificial corneas has been dramatically increased. For instance, fewer than 50 units of B-KPro were implanted before 2002, while more than 9,000 implantations were carried out in 2014. In this section, recent advances in the design of artificial corneas will be discussed with regard to the type of materials employed.

The B-KPro is the most widely implanted artificial cornea and was developed at the Massachusetts Eye and Ear Infirmary. There are two types of B-KPro each with different indications. The B-KPro type I is commonly used in patients with a non-cicatrizing disease such as repeated allograft failure, corneal opacity with extensive neovascularization, aniridia, trauma, etc. In contrast, B-KPro type II is used in cicatrizing diseases and severe dry eye conditions, such as severe autoimmune ocular diseases. Both types of B-KPro have similar compartments but are different in some details. They have a front plate and a back plate, which act to sandwich a fresh donor cornea; a titanium locking-ring is used to secure the plates. Medical grade polymethyl methacrylate (PMMA) with the ability to block UVA/UVB is used for the front and back plates. The idea of using PMMA was conceived due to its ability to induce only minimal inflammatory responses in the eye (Griffith et al., 2016). PMMA is a transparent thermoplastic polymer, also known as acrylic glass, which can be modified to achieve desired mechanical properties, like toughness and stiffness. On the other hand, modifying the PMMA polymer with nano-dimensional TiO2, SiO2, ZnO, ZrO2, Al2O3, CNT, and graphene has been investigated for electromagnetic shielding, thermal insulation, antiglare resistance, scratch resistance, and also resistance against UV radiation (Pandey et al., 2010; Cano et al., 2013; Soumya et al., 2014). The apparent success of PMMA in keratoprostheses has been plagued by numerous complications like tissue necrosis, retroprosthetic membrane formation, vitreous opacities, etc. The main reasons for these shortcomings include poor adhesion between the PMMA and the host corneal collagen at the PMMA-host interface and fibrous membrane formation due to fibroblast attachment onto the inner surface of PMMA. Therefore, there have been ongoing research efforts to overcome these drawbacks. In the first attempts, PMMA surfaces were modified with polyethylene glycol (PEG) or extracellular matrix molecules to block cell adhesion (Kim et al., 2001; Aucoin et al., 2002). The results were promising in terms of preserving the inner surface of PMMA but, at the external PMMA-host interface, the reduction in cell attachment resulted in endophthalmitis and extrusion of the implant. Endophthalmitis is an internal inflammation of the eye, which is a possible complication of B-KPro, arising because of inadequate integration of PMMA with the host corneal tissue allowing penetration of microorganisms. Suggested ways to reduce this drawback include using of daily antibiotics and modifying the B-KPro surface. Using antibiotics is out of the scope of this paper, so only surface modification of PMMA will be discussed.

Surface modifications of PMMA have been studied using two approaches. One of them focuses on improving cell attachment at the PMMA-corneal tissue interface to prevent the penetration of microorganisms into the internal part of the eye. In this context, Patel et al. (2006) studied the region-specific control of cell adhesion by grafting di-amino-PEG onto the surface of PMMA which could be used to improve cell adhesion and spatially control cell attachment onto the PMMA. Recently the

membrane, is included for completion. The macroscopic, microscopic, and nanoscopic features are emphasized (from left to right) to help illustrate the various interactions between the tissue components. Bowman's layer is essentially a random fibril, woven-mat composite, which maximizes multi-axial stiffness and strength. The underlying anterior third of the stroma proper is a lamellar interwoven fabric composed of unidirectionally (UD) fibril-reinforced lamellae. This architectural hierarchy is more rigid against z-axis deformations compared to non-woven UD-laminates. In the human body, the corneal structure is most similar to that of the pericardium, which serves to mechanically prevent the formation of aneurysms in the heart. The posterior two-thirds of the stroma is essentially a non-woven, UD-fibril-reinforced lamellar composite, which maximizes longitudinal x- and y-axis stiffness and strength, but has only weak transverse z-axis stiffness and strength. In the human body, its structure is most similar to that of the annulus fibrosis of the intervertebral disk, which functions efficiently as a cushioning mechanism for the spine, but is prone to chronic biomechanical failure. The UD-orientation of collagen fibrils in each lamella is vital because this arrangement prevents fibril undulation and thus maximizes the initial axial tensile strength of each fibril. Descemet's membrane forms a hexagonal lattice. Taken together these composite-like regions are responsible for the overall stiffness, strength, extensibility, and toughness of the cornea. They also help explain how the cornea behaves biomechanically after surgery, disease, or injury. Reprinted with permission (Kaufman et al., 2011).

stability and biocompatibility of poly (2-hydroxyl methacrylate) polymethyl methacrylate (PHEMA-PMMA) has been studied and has shown promising optical transparency, flexibility, and good mechanical properties. Nevertheless, its stability and biocompatibility still need further research (Hwang and Kim, 2016). The second approach is to fabricate a PMMA polymer with antibacterial properties. Examples of these antibacterial coatings are titanium dioxide (TiO2) (Riau et al., 2016) and silver nanocluster (Baino et al., 2016). For instance, Salvador-Culla et al. showed that a TiO2 coating on PMMA enhanced keratocyte cell integration and attachment, while at the same time demonstrated antibacterial properties (Salvador-Culla et al., 2016). In another attempt, researchers at the University of California, Irvine (UC Irvine) mimicked the surface of insect wings to make an antibacterial PMMA for using in an artificial cornea (Kowalski, 2016).

In the presence of severe ocular surface inflammation like chemical burns, end-stage Stevens-Johnson syndrome, ocular cicatricial pemphigoid, multiple failed penetrating keratoplasties, and trachoma, the use of the Osteo-Odonto-Keratoprosthesis (OOKP) is an alternative approach. Multiple efforts in using more biologically compatible substances resulted in the introduction of OOKP in 1963 by Strampelli (Han et al., 2015b). In this kind of artificial cornea, an autologous tooth was cut horizontally (OOKP) or longitudinally (MOOKP) to allow an optical cylinder made of PMMA to be introduced inside it. MOOKP is a modified OOKP introduced by Falcinelli et al. (1987). Tibia KPro is another type of biological keratoprosthesis in which a fragment of the autologous tibia was used to allow anatomical retention for an extended period of time. The biological parts of OOKP and MOOKP are known as a "keratoprosthesis skirt" and various studies have suggested that the use of living materials results in lowering the risk of extrusion and infection (Falcinelli et al., 2005). Beside the biological parts of OOKP and MOOKP, two other types of synthetic materials are used in these implants. These are (a) the transparent material, which is used in the optical cylinder, and (b) the adhesive, which is used to bond the optical cylinder to the biological parts. PMMA is a gold standard transparent, water-resistant, and durable material for the optical cylinder. However, as mentioned before, there are ongoing studies toward improving its characteristics. Acrylic bone cement is a standard dental adhesive, which is commonly used, and its suitability has never been seriously challenged. Yet, studies on other dental adhesives like glass ionomer and universal resin cement have been conducted (Weisshuhn et al., 2014; Alarcon et al., 2016).

In comparison with B-KPo, MOOKP is cheaper and does not require a viable donor cornea. However, there are some obstacles and controversial issues related to the use of OOKP and MOOKP. For instance, two fundamental surgical procedures need to be performed to implant the biological keratoprosthesis, and there is a 3-month interval between these procedures. This requirement complicates the surgery and reduces patient satisfaction. As a result, in the last decade, research on OOKP and MOOKP has focused on replacing the biological skirt with a synthetic skirt that enables it to biointegrate with the surrounding corneal tissue. Synthetic skirts have shown better mechanical biocompatibility with the sclera, in addition to more comfortable fabrication and better handling during the surgery. AlphaCor is one of the commercially available artificial corneas with a synthetic skirt. This kind of keratoprosthesis has been studied and developed in the Lion Eye Institute of Western Australia since 1989. AlphaCor is a one-piece device with two concentric regions that made from poly-(2-hydroxyethylmethacrylate) (PHEMA). The central core of the device is a transparent PHEMA cylinder, and the skirt is a porous PHEMA material to improve biointegration with the surrounding corneal stromal tissue. Nevertheless, the rejection rates of the synthetic skirt is much higher than the biological skirts discussed above (Polisetti et al., 2013). Thus, improving the biointegration of the synthetic skirt is an important objective in recent studies. In this regard, Pino et al. (2008) attempted to increase the bioactivity of various synthetic polymers using biomimetic coatings. They suggested that growing a bioactive apatite layer on the surface of the polymers would provide well in vivo biointegration. Another approach to enhancing the bioactivity of the skirt is to modify the surface of the skirt with extracellular matrix proteins like fibronectin, laminin, and collagen (Xie et al., 1997). Moreover, replacing the polymers with bioactive materials like bioglass has also been investigated (Laattala et al., 2011). Accordingly, Huhtinen et al. (2013) replaced the polymeric skirt of the keratoprosthesis with bioactive glass. They claimed that the porous bioglass had a capacity to induce and support tissue ingrowth, resulting in better biointegration. Similar investigations using other innovative materials have been proposed by other research groups. Recently, Tan et al. (2015) assessed the potential of two-dimensional graphene film and 3D graphene foam as a next-generation biomaterial for the synthetic keratoprosthesis skirt.

Considering the advantages and disadvantages of the three commercial keratoprosthesis which have been discussed above, KeraMed Inc. (Sunnyvale, California) has introduced a newer design to address the limitations of previous keratoprostheses while retaining their advantages. KeraKlear Artificial Cornea is a one-piece keratoprosthesis without any need for a donor cornea. Moreover, using only acrylic material allows KeraKlear to be foldable and injectable. However, its implantation is technically challenging, and further investigations are needed to overcome these challenges (Pineda, 2015). Besides, KeraKlear is a new product, and more clinical evaluation is necessary to ensure its safety and efficacy. To summarize the above discussions, the keratoprostheses are compared with two other recently introduced artificial corneas in **Table 1** and **Figure 3**.

# CORNEAL WOUND HEALING

Corneal wound healing, like wound healing in general, is a complex and dynamic process which is divided into four phases: the hemostasis, inflammation, cell proliferation, and remodeling phases. In fact, wound healing involves the interaction of various different cell lineages and a choreographed series of cellular events resulting in the replacement of the missing tissue or cellular structures. The cornea has special characteristics, which are important to consider in choosing a treatment strategy for wound healing. The most prominent characteristics of the cornea include its limbus zone, its lack of blood vessels, and its immune privilege (see **Figure 4**). As a result, current studies in corneal wound healing treatment have focused on a corneal characteristic like an immune response, avoiding angiogenesis and modulating cell signaling. In this section, topics related to angiogenesis and immune privilege, such as blocking immune signaling pathways, exosomes, biopolymers, growth factors, and use of amniotic membrane, will be discussed. New approaches to corneal wound healing related to the limbus zone will be reviewed in the following section (Simpson et al., 2019).

# Corneal Immune Privilege and Avoidance of Angiogenesis

Generally, the transparency and avascularity of the cornea are essential for proper vision. The main factors, which are threatening the corneal clarity are inflammatory reactions, neovascularization, and limbal deficiency. As a result, avoiding angiogenesis, maintaining immune privilege, and supporting the limbus zone are strategies that have been investigated to combat processes that endanger corneal transparency. Immune privilege describes the natural lack of inflammation in the cornea, but when wound healing is taking place, the immune response needs to be modulated and limited. The influx of inflammatory cells causes scar formation and destruction of the limbus zone. Since there is a naturally enhanced production of proangiogenic factors during wound healing (Cursiefen, 2007; Ellenberg et al., 2010),

\*Moscow eye microsurgery complex in Russia.

limbus deficiency endanger corneal transparency. Reprinted with permission from Ellenberg et al. (2010) and Haagdorens et al. (2016).

actions need to be taken to restrict the undesirable growth of blood vessels into the cornea. In the following section, recent studies on this issue will be discussed.

### Blocking Immune Signaling Pathways