Preparation and Evaluation of Thiol-Modified Gelatin Nanoparticles for

Bioconjugate Chem. 2005, 16, 1423−1432

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Preparation and Evaluation of Thiol-Modified Gelatin Nanoparticles for Intracellular DNA Delivery in Response to Glutathione Sushma Kommareddy and Mansoor Amiji* Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, 110 Mugar Life Sciences Building, Boston, Massachusetts 02115. Received May 20, 2005; Revised Manuscript Received August 18, 2005

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we have developed thiolated gelatin nanoparticles that can release the payload in the highly reducing environment, such as in response to glutathione. Thiolated gelatin was synthesized by covalent modification of the primary amino groups of Type B gelatin using 2-iminothiolane (Traut’s reagent). The degree of thiolation of the polymers ranged from 0 to 43.71 mmol of reduced sulfhydryl (SH) groups when the amount of 2-iminothiolane was increased up to 100 mg per gram of the biopolymer. Cytotoxicity evaluations carried out by the formazan (MTS) assay showed that the thiolated gelatin prepared with 20 mg and 40 mg of 2-iminothiolane (SHGel-20 and SHGel-40) per gram of gelatin had comparable cell viability profile to that of the unmodified gelatin. In vitro release studies of fluorescein isothiocyanate (FITC)-labeled dextran (mol wt. 70 000 Da), when encapsulated in gelatin and thiolated gelatin nanoparticles (150-250 nm in diameter), was found to be affected by the presence of glutathione (GSH) in the medium. The presence of GSH was found to enhance the release by about 40% in case of thiolated gelatin and about 20% in gelatin nanoparticles under similar conditions of temperature and GSH concentrations. Qualitative and quantitative analysis of transfection in NIH-3T3 murine fibroblast cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by fluorescence confocal microscopy and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 h. Quantitative results from FACS showed that the thiolated gelatin nanoparticles (SHGel-20) were significantly more effective in transfecting NIH-3T3 cells than other carrier systems examined. The results of this study show that thiolated gelatin nanoparticles would serve as a biocompatible intracellular delivery system that can release the payload in a highly reducing environment.

INTRODUCTION

In recent years, there has been an enormous interest in ex vivo and in vivo gene therapy approaches, which necessitates the formulation of DNA into vectors for targeting a specific population of cells either locally or systemically. The gene delivery to target cells can be achieved by either viral or nonviral methods (1-6). Viral vectors, although efficient in transfection, are plagued by issues of integration with the host genome, selfreplication, recombination potential, and immunogenicity. There are several reported incidents of insertional mutagenesis with viral vector administration in humans leading to serious safety concerns (7-9). Viral vectors are also relatively expensive to manufacture and have difficulties in bypassing the immune defense mechanism. The nonviral vectors for gene delivery applications are increasingly popular owing to several advantages, such as ease of production and administration and lack of immunogenicity and mutagenesis. An ideal vector used in gene therapy needs to be inert while in circulation and release its payload at the target site resulting in an efficient transfection of the cells. The vector used should have sufficiently small size and stability, minimal aggregation in blood, and the ability to efficiently target cells. Once in the cell, the system should disassemble and release intact DNA in the supercoiled state for nuclear uptake and transcription. Cationic lipids and biodegrad* Corresponding author: Tel. (617) 373-3137. Fax (617) 3738886. E-mail: [email protected].

able polymers have been synthesized to condense DNA and promote efficient intracellular delivery (10). However, the design of an ideal vector is still a limiting step, and various strategies are needed for achieving effective nonviral gene therapy (11). Polymeric nanoparticles offer an attractive alternative for intracellular gene delivery because of their high surface-to-volume ratio and their ability to encapsulate DNA without a precondensing step (12). In addition, these colloidal carriers are stable and can be prepared and modified with relative ease. Tissue- and cell-targeted polymeric nanocarriers can be obtained by attaching specific ligands such as antibodies or lectins in order to achieve site-specific delivery, and the surface can be modified with hydrophilic polymers, such as poly(ethylene glycol) (PEG), resulting in long circulating properties in the systemic circulation. Due to the small size these nanoparticles can be used to achieve tumor targeting by the enhanced permeability and retention (EPR) effect (13-15). For the past several years, our group has been engaged in exploring the use of gelatin and modified gelatin-based nanoparticles for intracellular drug and gene delivery. The nanoparticulate carriers of gelatin have been used for efficient intracellular delivery of the encapsulated payload. Kaul and Amiji (16, 17) have shown that Type-B gelatin and PEG-modified gelatin nanoparticles loaded with TMR-dextran, as a model hydrophilic macromolecule, can be used for cell trafficking (BT-20 cells). Also, from the trafficking experiments, using colloidal gold-

10.1021/bc050146t CCC: $30.25 © 2005 American Chemical Society Published on Web 09/28/2005

1424 Bioconjugate Chem., Vol. 16, No. 6, 2005

encapsulated gelatin nanoparticles, it was evident that nanoparticles were efficiently taken up by the cells through nonspecific endocytosis and accumulated in the perinuclear region. Furthermore, it was reported that plasmid-encapsulated gelatin and PEG-modified gelatin nanoparticles were internalized in NIH-3T3 fibroblast cells within the first 6 h of incubation, and the transfection results are quantified by flow cytometry at different time points up to 96 h (18). Kaul and Amiji also showed the biodistribution and tumor targeting results of gelatin and PEG-modified gelatin nanoparticles, labeled with iodine-125, upon intravenous administration in mice bearing Lewis lung carcinoma (LLC) (19). From the radioactivity in plasma, tumor mass, and various organs collected, it was evident that the majority of PEGylated nanoparticles were present either in the blood pool or taken up by the tumor mass and liver. The plasma and the tumor half-lives, the mean residence time, and the area-under-the-curve of the PEGylated gelatin nanoparticles were significantly higher than those for the gelatin nanoparticles. The results of this study showed that PEGylated gelatin nanoparticles do possess long circulating properties and can preferentially distribute in the tumor mass after systemic delivery (19). Last, it has been shown that the in vivo transgene expression in LLC tumor bearing C57BL/6J mice administered with plasmid DNA encoding β-galactosidase (pCMV-β) encapsulated in gelatin and PEG-modified gelatin nanoparticles. Qualitative and quantitative analysis has shown a significantly greater transfection efficiency with the use of PEGmodified gelatin nanoparticles following systemic administration (20). It is the unique physical, chemical, and biological properties of the gelatin and its modified derivatives that make this system suitable for systemic gene delivery and other therapeutic applications (16, 18, 19, 21-26). Despite numerous advantages of gelatin, it is still sometimes necessary to chemically modify the biopolymer so that it remains stable during blood circulation which can lead to efficient expression at the target site. Based on the fact that the free thiol groups can form disulfide bonds within the polymer, the added thiol groups can be used in strengthening the tertiary and quaternary protein structure. The disulfide bonds also stabilize the nanoparticles, and when they are broken, the polymer unfolds, releasing the encapsulated payload. This mechanism is environment-sensitive and is enhanced by the presence of glutathione (GSH) or other redox enzymes that are present in high concentrations in the hypoxic regions, such as the tumor cells. The cells generally contain majority of the glutathione in the cytosol and at concentrations ranging from 1 to 11 mM, while the extracellular concentration is about 1000-fold less (2729). On the basis of the differences in the intracellular and extracellular GSH concentrations, we hypothesize that thiolated gelatin nanoparticles will provide a unique platform for intracellular DNA delivery in response to GSH levels. In the present study, therefore, we describe the synthesis of thiolated gelatin and preparation of nanoparticulate systems for intracellular plasmid DNA delivery. Transgene expression efficiencies, using enhanced green fluorescent protein (EGFP-N1) plasmid, were examined by qualitative and quantitative analysis. EXPERMENTAL PROCEDURES

Materials. Type-B gelatin (225 bloomstrength) with 100-115 mmol of carboxylic acid per 100 g of protein,

Kommareddy and Amiji

an isoelectric point of 4.7-5.2, and an average molecular weight 40 000-50000 Da was purchased from SigmaAldrich (St Louis, MO). 2-Iminothiolane (Traut’s reagent) and 5,5′- dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) were purchased from Pierce Biotechnology Inc., (Rockford, IL). CellTiter 96 AQueous one solution cell proliferation assay was obtained from Promega Corporation (Madison, WI). Ethyl alcohol, absolute, 200 proof, 99.5% ACS reagent, was obtained from Acros Chemicals (Pittsburgh, PA). Glyoxal 40% w/v and glycine were purchased from Fisher Scientific (Fair Lawn, NJ). Poly(ethyleneimine) (PEI, Mn ) 10 000 Da) and fluorescein isothiocyanate (FITC) dextran with a molecular weight 70 000 Da and degree of substitution of 0.004 mol of FITC per mole of dextran were obtained from Sigma as well. The NIH3T3, mouse embryonic fibroblast cell line, was obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco’s modified Eagle medium (DMEM) with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose and supplemented with 10% fetal bovine serum and 1% each of penicillinstreptomycin, HEPES buffer, and sodium pyruvate. General purpose serum-free medium (UltraCulture) was obtained from BioWhittaker (Walkersville, MD). Lipofectin lipid transfection reagent was obtained from Invitrogen (Carlsbad, CA). All aqueous solutions and reagents were prepared in deionized distilled water from Nanopure II (Barnstead/Thermolyne, Dubuque, IA). Synthesis and Purification of Thiolated Gelatin. Thiolated gelatin was synthesized by covalent modification of the primary amino groups of gelatin by the addition of sulfhydryl moieties, and the chemistry involved in thiolation is shown in Figure 1. For the synthesis, 1 g of gelatin was dissolved in 100 mL of deionized water and then incubated with varying amount of 2-iminothiolane hydrochloride, 20 mg (SHGel-20), 40 mg (SHGel-40), and 100 mg (SHGel-100) at room temperature for 15 h (30). Any unreacted 2-iminothiolane was removed by repeated dialysis against 5 mM HCl followed by 1 mM HCl solution for 24 h each. The purified thiolated gelatin was dried in vacuo and stored at -80 °C for further use. Determination of the Degree of Thiolation. To determine the degree of thiolation, the control gelatin and thiolated gelatin derivatives were dissolved in 0.1 M sodium phosphate buffer (pH 8.0) containing 1 mM EDTA. The colorimetric reaction was carried out by mixing 500 µL of the sample solutions with 100 µL of Ellman’s reagent (4 mg/mL) and 5 mL of buffer. After 15 min of reaction at room temperature, the absorbance of the solution was measured at 412 nm by Shimadzu UV160U spectrophotometer (Columbia, MD) (31). The degree of thiolation was calculated by extrapolating the results obtained to that of the standard curve of cysteine and expressed in terms of millimoles of free sulfhydryl groups per gram of gelatin. Preparation of Nanoparticles. Nanoparticles of the control gelatin and thiolated gelatin derivatives were prepared by desolvation using ethanol under controlled conditions of temperature and pH as previously described (16). Briefly, a 1% (w/v) solution in water was prepared by dissolving 200 mg of the control or thiolated gelatin in a temperature controlled water bath at 37 °C. The pH of the resulting solution was adjusted to 7.0 with 0.2 M sodium hydroxide. Nanoparticles were formed when the solvent composition was changed from 100% water to 75 vol % hydro-alcoholic solution, upon gradual addition of ethanol, under continuous stirring conditions. The formed nanoparticles were further cross-linked with 0.1 mL of

Thiolated Gelatin Nanoparticles for Intracellular DNA Delivery

Bioconjugate Chem., Vol. 16, No. 6, 2005 1425

Figure 1. Schematic illustration of the synthesis of thiolated gelatin by reacting Type-B gelatin with 2-iminothiolane (Traut’s Reagent).

40% (v/v) aqueous solution of glyoxal for desired time interval, and any unreacted aldehyde was quenched with 0.2 M glycine solution. The particles obtained were centrifuged either before or after cross-linking at 16 000 rpm for 30 min. The nanoparticles were washed twice with deionized distilled water and lyophilized. Characterization of Nanoparticles. Particle Size Analysis. The resulting non-cross-linked and cross-linked systems of the control and thiolated gelatin nanoparticles were analyzed for mean particle size by ZetaPALS, 90Plus (Brookhaven Instruments Corporation, Holtsville, NY). The colloidal suspension of the nanoparticles was diluted with deionized distilled water, and the particle size analysis was carried out at a scattering angle of 90° and a temperature of 25 °C. Scanning Electron Microscopy. The gelatin and thiolated gelatin nanoparticles were separated from suspension by centrifugation at 16 000 rpm for 30 min and lyophilized. The freeze-dried nanoparticles were mounted on an aluminum sample mount and sputter-coated with gold-palladium to enhance conductivity and minimize the buildup of charges. The samples were then observed for surface morphology under a Hitachi 4800 field emission scanning electron microscope. Surface Charge Measurements. Zeta potential (ξ) measurements of the control and thiolated gelatin nanoparticles (both non-cross-linked and cross-linked systems) in suspension in deionized water were performed using a ZetaPALS (Brookhaven Instruments Corporation, Holtsville, NY). The nanoparticles were dispersed in deionized water and the zeta potential values measured at the default parameters of dielectric constant, refractive index, and viscosity of water.

Cell Viability Studies with Thiolated Gelatin. The NIH-3T3 murine fibroblast cells were grown in 96-well plates at an initial seeding density of 10 000 cells per well in 150 µL of supplemented DMEM. The cells were allowed to grow for 24 h, and the growth medium was replaced by serum free media containing gelatin and thiolated gelatins in aqueous solutions at a concentrations ranging from 0 to 200 µg/mL along with negative control (serum free media alone). PEI, a known cytotoxic cationic polymer, was used as a positive control. The cells were treated with the control and test materials for 6 h and then replaced by 20 µL of MTS along with 100 µL of culture media per well. The cells were incubated for ∼2 h along with the reagent at 37 °C in a 5% CO2 incubator, and the absorbance of the purple colored formazan product formed was measured at 490 nm with a Biotek SynergyHT plate reader (Winooski, VT). The percent viability of the cells was expressed as the ratio of absorbance of the polymer-treated cells relative to those in serum alone multiplied by 100 and plotted as a function of the polymer concentration. Glutathione-Responsive In Vitro Release Studies. FITC-dextran of molecular weight 70 000 Da with a degree of substitution of 0.004 mol of FITC per mole of dextran was used as a model of hydrophilic macromolecular drugs. A 1.0% (w/v) solution of gelatin and thiolated gelatins (SHGel-20 and SHGel-40) in water were prepared and adjusted to pH 7.0, followed by addition of FITC-dextran at a concentration of 1.0% (w/ w) to the polymer. Using this solution FITC-dextran loaded nanoparticles were prepared by desolvation process as described above. The free FITC-dextran was separated from the loaded by a series of centrifugation


and washing steps, and the loading efficiency in the nanoparticulate formulations was determined by dissolving the nanoparticles in PBS containing protease (0.2 mg/ mL) at 37 °C. The resulting solution was filtered through a 0.22 µm filter, and the fluorescence intensity was measured at 490 nm excitation and 520 nm emission wavelengths, using a Biotek SynergyHT plate reader (Winooski, VT). The efficiency of FITC-dextran loading in the nanoparticle formulations was determined using a standard curve of FITC-dextran in protease-containing PBS. The in vitro release of FITC-dextran from the nanoparticles was performed in varying concentrations of glutathione (GSH) in PBS at 37 °C and compared with the release in PBS alone. The study was carried out in PBS and 0.1, 1.0, and 5.0 mM GSH containing PBS at 37 °C. Twenty milligrams of the FITC-dextran-loaded control gelatin and thiolated gelatin nanoparticulate samples were weighed into microcentrifuge tubes with 1.5 mL of the buffer solutions. The samples were placed in a temperature-controlled bath, and 0.5 mL of the supernatant was removed at specified intervals following centrifugation at 10 000 rpm for 5 min. Sink conditions were maintained by replacing an equal volume of the release medium each time. The fluorescence intensity of the released FITC-dextran in the samples was measured using a plate reader, and the percentage of dye released was calculated from calibration curves constructed in PBS containing various concentrations of glutathione. The release studies were repeated for both cross-linked and non-cross-linked systems. Preparation and Characterization of Plasmid DNA-Encapsulated Nanoparticles. Nanoparticles encapsulating plasmid DNA expressing enhanced green fluorescent protein (EGFP-N1) were prepared by desolvation using ethanol under controlled conditions of temperature and pH as previously described. The pH of the 1% (w/v) solution of control gelatin or thiolated gelatin (SHGel-20, SHGel-40, and SHGel-100) was adjusted to 7.0, and 1 mL of the plasmid DNA at a concentration of 1 mg/mL was added. The nanoparticles encapsulated with plasmid DNA were formed by controlled precipitation of the aqueous gelatin solution with ethanol under continuous stirring. The nanoparticles were centrifuged at 16 000 rpm for 30 min either before or after cross-linking and were analyzed for mean particle size, size distribution, and zeta potential by ZetaPALS (Brookhaven Instruments Corporation, Holtsville, NY). DNA Encapsulation Efficiency. The pDNA-loaded nanoparticles were separated from the free pDNA by a series of centrifugation and washing steps. The loading efficiency of pDNA in the nanoparticle formulations was determined by dissolving the nanoparticle sample in protease (0.2 mg/mL)-containing PBS at 37 °C. The pDNA concentrations were estimated in solution using the PicoGreen dsDNA reagent (Molecular Probes, Eugene, OR), a one step, sensitive, fluorescent, quantitation kit for double-stranded DNA (dsDNA). The reagent was incubated with the samples for 5 min, and the fluorescence intensity was measured at 480 nm excitation and 520 nm emission wavelengths, using a Bio-Tek plate reader. Stability of Encapsulated Plasmid DNA. The stability of the encapsulated plasmid DNA was confirmed by running the extracted DNA on precast gels. The nanoparticles were digested using protease containing PBS, diluted, and mixed with 5-fold diluted loading buffer (1×). The samples were then loaded onto 1.2% agarose gels (E-Gel, Invitrogen, Carlsbad, CA) prestained with


ethidium bromide at a concentration of 100 ng/well in a 15 µL volume per well. Naked plasmid DNA and DNA encapsulated in gelatin nanoparticles were used as control and the gel was run at 65 V for 30 min using a Biorad model 200/2 power supply, and the bands were visualized using KODAK Gel logic 100 imaging system (Scientific Imaging, New Haven, CT). The process was repeated for both cross-linked and non-cross-linked nanoparticles. In Vitro Transfection Studies. Qualitative Analysis of Transfection. NIH-3T3 cells, murine fibroblast cells, were grown in six-well cell culture plates containing a Corning’s circular glass cover-slip with 2 × 105 cells seeded per well. The cells were grown to semi-confluence in an incubator at 37 °C and 5% CO2 atmosphere. The plasmid DNA encapsulated non-cross-linked and crosslinked nanoparticles prepared from control gelatin, and thiolated gelatins were dispersed in serum-free medium. Twenty micrograms of EGFP-N1 plasmid DNA complexed with 20 µL of Lipofectin, a cationic lipid transfection reagent, was used as a positive control and untreated cells were used as a negative control. After filtration, the DNA-containing nanoparticle suspension was added at a concentration equivalent to 20 µg of plasmid DNA per well and incubated with the cells at 37 °C for a period of 6 h. The media was removed from each well and replaced with the DMEM. The cover slips were washed three times with sterile PBS at time intervals of 12, 24, 48, and 96 h postincubation and mounted on to microscopic slides containing a drop of fluorescence-free mounting medium (Fluoromount-G, Southern Biotech Associates, Birmingham, AL). The expression of GFP in the cells was observed by a fluorescence microscope. Differential interference contrast (DIC) and fluorescence images acquired using Olympus BX61 microscope and the digital images were processed with Adobe Photoshop software. Quantitative Analysis of Transfection. NIH3T3 cells were grown to semi-confluence in a six-well cell culture plate and transfected with EGFP-N1 plasmid DNA encapsulated non-cross-linked and cross-linked systems of gelatin, SHGel-20, and SHGel-40 nanoparticles and compared with the Lipofectin-plasmid DNA transfected cells and cells treated with serum free media alone. The nanoparticles carrying pDNA and the Lipofectin-DNA mixture were dispersed in serum free media and added to each of the six wells at a concentration of 20 µg of plasmid DNA per well. The cells are allowed to incubate for a period of 6 h at 37 °C in a 5% CO2 incubator and then replaced by fresh growth medium. The adherent cells are trypsinized at 12, 24, and 48, and 96 h postincubation, centrifuged, and resuspended in 0.5 mL of DMEM before fixing with 100 µL of 4% formalin buffer solution. The fluorescence of the GFP produced in the transfected a cell was detected by a Beckman Coulter (Epics/Altra) (FACS) equipped with an argon 488 laser. The FL1 channel was used to detect the cells expressing GFP fluorescence, and the results obtained were analyzed using Expo 32 software. RESULTS AND DISCUSSION

Synthesis and Characterization of Thiolated Gelatin. The degree of thiolation was calculated by determining the number of free sulfhydryl groups using Ellman’s reagent. The results obtained were extrapolated to that of the standard curve of cysteine and expressed in terms of millimoles of free sulfhydryl groups per gram of the polymer and are shown in Table 1. There was an increase


Thiolated Gelatin Nanoparticles for Intracellular DNA Delivery Table 1. Characterization of the Control and Thiolated Gelatin Nanoparticles amount of iminothiolane (mg) per gram of gelatin

degree of thiolation (mM/g gelatin)

0 mg (gelatin) 20 mg (SHGel-20) 40 mg (SHGel-40) 100 mg (SHGel-100)

0 6.08 ( 0.7b 16.41 ( 0.14 43.71 ( 0.35

a

mean diameter of nanoparticles (nm) non-cross-linked cross-linked 151.3 ( 53.8a 156.6 ( 60.7 165.3 ( 45.0 150.9 ( 58.4

230 ( 11.5 244 ( 6.8 232.8 ( 6.4 253.7 ( 8.9

zeta potential (mV) non-cross-linked cross-linked -8.86 ( 0.78 -9.09 ( 0.81 -9.83 ( 0.74 -11.97 ( 1.20

-8.45 ( 0.45 -9.36 ( 0.64 -11.88 ( 0.72 -12.98 ( 0.77

Mean ( SE (n ) 8). b Mean ( SD (n ) 4).

in the degree of thiolation with increasing amounts of iminothiolane, with SHGel-20 containing 6.08 mmol/g followed by SHGel-40 (16.4 mmol/g) and SHGel-100 (43.7 mmol/g). The increasing amounts of iminothiolane resulted in an increase in thiolation at the free amino groups present on gelatin. Preparation and Characterization of the Control and Thiolated Gelatin Nanoparticles. The nanoparticles prepared by desolvation were characterized for particle size and zeta potential. The average size and surface charge of the particles prepared from thiolated gelatins with different degrees of thiolation are listed in Table 1. The mean particle size for both non-cross-linked control and thiolated gelatin nanoparticles was found to be around 150 nm and that of cross-linked nanoparticles at 200 nm. The net charge on the gelatin nanoparticles was found to be negative, and there was no significant difference in the values between the cross-linked and non-cross-linked nanoparticles. The control gelatin nanoparticles were found to have a zeta potential value of -8.86 mV as compared to the -11.97 mV for the thiolated gelatin (SHGel-100) nanoparticles. The slight difference in zeta potential values between control and thiolated gelatin nanoparticles could be due to the fact that a hydrochloride salt of 2-iminothiolane was used. The surface presence of chloride ions would result in the nanoparticles having a greater negative zeta potential value with increasing amount of 2-iminothiolane used. The net negative charge on the surface adds to the stability of these colloidal particles. On the basis of these results, we find that the solvent exchange method can still be used to make nanoparticles from thiolated gelatin with different degrees of thiolation. Also, from these studies, we find that there was no significant difference in the particle size of the formed nanoparticles between gelatin and thiolated gelatin. Cross-linking of the nanoparticles did increase the size, probably due to disulfide bridging of two or more nanoparticles. Lastly, the surface charge on nanoparticles was also not affected by the degree of thiolation. SEM images shown in Figure 2 reveal the surface morphology and spherical shape of the control and thiolated gelatin nanoparticles. The actual diameter of the nanoparticles observed by SEM was around 200 nm and was found to be similar to the values obtained by measurements based on light scattering. From the studies of Jain and co-workers, it is known that molecular size is one of the key determinants of vascular transport in tumors and that tumor vessels are permeable and have pores with a cutoff size of around 400-600 nm. Therefore, the thiolated gelatin nanoparticles with a mean particle size of around 200 nm would be effective in delivering DNA to solid tumors in vivo. The in vitro toxicity studies of the control and thiolated gelatins were conducted by MTS assay with NIH3T3 cells. The percent viability of the polymer-treated cells was compared with PEI (mol wt 10 kDa), a known cytotoxic DNA-condensing gene carrier, as a positive control. The results of the cell viability profile as seen in

Figure 2. Scanning electron micrographs of non-cross-linked gelatin (A) and thiolated gelatin nanoparticles prepared by reacting 20 mg of 2-iminothiolane per gram of gelatin (SHGel20) (B).

Figure 3. Cytotoxicity profiles of the control gelatin (9), and thiolated gelatin prepared with 20 mg of 2-iminothiolane (SHGel-20) (b), 40 mg of 2-iminothiolane (SHGel-40) ([), and 100 mg of 2-iminothiolane (SHGel-100) (2) per gram of gelatin. Poly(ethyleneimine) (mol wt 10 000 Da) (0) was used as a positive control. The relative cell viability was determined in NIH-3T3 murine fibroblast cells by the MTS assay and plotted against the polymer concentration in aqueous solution at 37 °C.

Figure 3 indicate that gelatin was highly biocompatible and nontoxic showing 100% viability at all concentrations tested. At the highest concentration of 200 µg/mL, SHGel20 showed 92% viability, SHGel-40 showed 88%, and SHGel-100 showed 79% relative cell viability. Increase in the amount of 2-iminothiolane used in thiolation did



Figure 4. Release profile of fluorescein isothiocyanate-labeled dextran (mol wt 70 000 Da) from non-cross-linked nanoparticles of control gelatin (b) and thiolated gelatin prepared with 20 mg of 2-iminothiolane (SHGel-20) (9) and 40 mg of 2-iminothiolane (SHGel40) (2) in phosphate-buffered saline (PBS, pH 7.4) (A) and in the presence of increasing glutathione (GSH) concentrations: 0.1 mM GSH (B), 1.0 mM GSH (C), and 5.0 mM GSH (D) containing PBS at 37 °C.

result in an increase in the cytotoxicity that has been observed from the relative cell viability profiles. Based on these cytotoxicity results, further studies were carried out with SHGel-20 and SHGel-40 nanoparticles only. In Vitro Release of FITC-Dextran in Response to Glutathione. Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) was used as a model for hydrophilic macromolecular drugs, as it can be encapsulated into nanoparticles by a simple process, impermeable to cellular membranes unless taken up by endocytosis and can be easily visualized under a florescence microscope. In addition, unlike other fluorophores such as rhodamine, the fluorescence of FITC is not affected by the presence of high glutathione concentration in solution. The loading efficiency was determined using FITC-dextran at a concentration of 1.0% w/w to that of the polymer. The non-cross-linked systems had a loading efficiency of 86.7% in case of nanoparticles prepared from gelatin followed by SHGel-20 and SHGel-40 with a loading efficiency of 91.9% and 99.7%, respectively. A similar trend was observed in loading efficiencies of cross-linked systems with gelatin nanoparticles having 84.6% followed by SHGel-20 with 86.1% and SHGel-40 with 99.6%. The in vitro release of FITC-dextran from the nanoparticles was performed in the presence of varying concentrations of GSH, which was included in the release medium to evaluate its nonenzymatic reducing action on the thiolated gelatin nanoparticles versus control gelatin nanoparticles. Also, we were interested in determining differences in the release behavior between non-crosslinked and cross-linked systems. As seen in Figures 4 and 5, the release of FITC-dextran from the non-cross-linked nanoparticles was significantly greater than from the cross-linked nanoparticles both in the presence and absence of glutathione in the release medium. The release from the thiolated gelatin nanoparticles was found to be faster in the presence of 5 mM GSH than in 1.0 mM GSH or 0.1 mM GSH containing PBS or PBS alone. In 5 mM GSH-containing PBS, the SHGel-20 nanoparticles released 100% of the payload content from the non-cross-

linked and 73.6% from the cross-linked system within 5 h. Similar results were obtained from SHGel-40 were 100% and 74% of FITC-dextran released from non-crosslinked and cross-linked nanoparticles in a span of 4 h, with cross-linked nanoparticles releasing up to 87.8% of their payload in 5 h. The presence of GSH was found to enhance the release by about 40% in case of thiolated gelatin and 20% in the case of gelatin nanoparticles. The predominant difference in the release characteristics can be attributed to the nonenzymatic reducing properties of GSH which cleaves off the disulfide bonds leaving the free thiol groups. Thus, the disulfide bonds formed within the thiolated gelatins could be used for achieving rapid release system in a highly reducing environment. Plasmid DNA Encapsulation in the Control and Thiolated Gelatin Nanoparticles. Nanoparticles encapsulating plasmid DNA expressing green fluorescent protein (pEGFP-N1) were prepared by desolvation using ethanol under controlled conditions of temperature and pH. The particles formed were harvested either before or after cross-linking and were characterized for particle size and zeta potential. Both the cross-linked and noncross-linked nanoparticles encapsulated with plasmid DNA were found to have mean particle size similar to that of the empty nanoparticles, with non-cross-linked control and thiolated gelatin nanoparticles around 150 nm and that of cross-linked nanoparticles at 200 nm. The zeta potential of the plasmid DNA encapsulated nanoparticles had values ranging from -9.01 for gelatin to -11.78. The net charge was found to be negative, and there was no significant difference in the values between the cross-linked and non-cross-linked nanoparticles as well as between the empty and DNA containing nanoparticles, indicating the encapsulation rather than adsorption of DNA on the surface of the particles. The loading capacity and efficiency of plasmid DNA in the nanoparticle formulations was determined using a standard curve of DNA in protease-containing PBS. The results as seen in Figure 6 indicate that cross-linking of gelatin does not affect the loading capacity of the nano-



Figure 5. Release profile of fluorescein isothiocyanate-labeled dextran (mol wt 70 000 Da) from cross-linked nanoparticles of control gelatin (b) and thiolated gelatin prepared with 20 mg of 2-iminothiolane (SHGel-20) (9) and 40 mg of 2-iminothiolane (SHGel-40) (2) in phosphate-buffered saline (PBS, pH 7.4) (A) increasing glutathione (GSH) concentrations: 0.1 mM GSH (B), 1.0 mM GSH (C), and 5.0 mM GSH (D) containing PBS at 37 °C.

Figure 7. Stability of encapsulated plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) by agarose gel electrophoresis. Gel electrophoresis of DNA encapsulated in noncross-linked (A) and cross-linked (B) nanoparticles. Lane 1 and 5 represent naked plasmid DNA (pEGFP-N1) as a control, lane 2 is DNA band from control gelatin nanoparticles, lane 3 is DNA band from thiolated gelatin nanoparticles prepared with 20 mg of 2-iminothiolane (SHGel-20), and lane 4 is DNA band from thiolated gelatin nanoparticles prepared with 40 mg of 2-iminothiolane (SHGel-40) per gram of gelatin. For the nanoparticle systems, the encapsulated plasmid was extracted by digesting the matrix in 0.2 mg/mL protease-containing phosphate-buffered saline (pH 7.4) at 37 °C.

Figure 6. The encapsulation of plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) in control gelatin and thiolated gelatin nanoparticles. Loading studies of pDNA (pEGFP-N1) from non-cross-linked (0) and cross-linked (9) nanoparticles prepared using control gelatin and thiolated gelatin prepared with 20 mg of 2-iminothiolane (SHGel-20) and 40 mg of 2-iminothiolane (SHGel-40) per gram of gelatin. The DNA encapsulation was expressed in terms of capacity as µg of DNA loaded per mg of nanoparticles (A) and efficiency as percent encapsulated (B). DNA encapsulation was determined by digesting the nanoparticles in the presence of 0.2 mg/mL protease-containing in phosphate buffered saline at 37 °C.

particulate systems. The gelatin nanoparticles had a loading efficiency of 99% as compared to SHGel-20, which

had the loading efficiency of 94%, and SHGel-40, which had the loading efficiency of 90%. The slight decrease in DNA loading with an increase in the degree of thiolation could be explained by the fact that DNA is negatively charged, and with increase in the negative charge of the thiolated gelatin there was a slight decrease in encapsulation efficiency which could be attributed to electrostatic repulsion. Stability of Encapsulated Plasmid DNA. The effect of thiolated gelatin and process parameters on the stability of DNA was detected by gel electrophoresis. The results, as seen in Figure 7, show that the plasmid DNA encapsulated in SHGel-20 and SHGel-40 are stable and are comparable to that encapsulated in gelatin nanopar-



Figure 8. Qualitative evaluation of enhanced green fluorescent protein (EGFP-N1) transgene expression in NIH-3T3 murine fibroblast cells with non-cross-linked control gelatin and thiolated gelatin nanoparticles prepared with 20 mg of 2-iminothiolane (SHGel-20) and 40 mg of 2-iminothiolane (SHGel-40) per gram of gelatin. Differential interference contrast (DIC) and fluorescence confocal images of the cells were obtained after 96 h upon incubation of DNA-containing nanoparticles with the cells in culture at 37 °C.

Figure 9. Qualitative evaluation of enhanced green fluorescent protein (EGFP-N1) transgene expression in NIH-3T3 murine fibroblast cells with cross-linked control gelatin and thiolated gelatin nanoparticles prepared with 20 mg of 2-iminothiolane (SHGel-20) and 40 mg of 2-iminothiolane (SHGel-40) per gram of gelatin. Differential interference contrast (DIC) and fluorescence confocal images of the cells were obtained after 96 h upon incubation of DNA-containing nanoparticles with the cells in culture at 37 °C.

ticles and naked plasmid DNA. The processes of crosslinking and protease digestion did not have any detectable effect on the DNA encapsulated and the polymers have a protective action on the plasmid. Qualitative and Quantitative Transgene Expression Studies. The qualitative analysis of GFP expression upon transfection in NIH3T3 cells was evaluated by florescence microscopy. The Figures 8 and 9 show the DIC and fluorescence images of cells transfected with cross-linked and non-cross-linked nanoparticles prepared from the control gelatin and thiolated gelatins. The images show that the polymeric nanoparticles carrying the plasmid DNA were able to transfect the cells and the protein expression was seen as early as 6 h (data not shown) and was stable for as long as 96 h posttransfec-

tion. The fluorescence images show that these nanoparticles protect the plasmid DNA during its transport which are later taken up by the cells through the endosomal pathway and released into the cytosol. The released DNA molecules would then diffuse into the nucleus through the nuclear pores and bring about transfection of the transgene. From the results it can also be noted that cross-linking and thiolation did not affect the ability of transfection of these plasmid DNA carrying nanoparticles. The quantitative analysis of transfection efficiency was carried out by flow cytometry. The transfection results at various time points for gelatin and the two thiolated gelatins were compared in non-cross-linked and crosslinked systems by plotting percentage of transfected cells



Figure 10. Quantitative evaluation of enhanced green fluorescent protein (EGFP-N1) transgene expression in NIH-3T3 murine fibroblast cells as a function of time as measured by flow cytometric analysis. Percent cell transfection with non-cross-linked (A) and cross-linked (B) nanoparticles encapsulated with EGFP-N1 plasmid DNA were prepared from control gelatin ([), and thiolated gelatin prepared with 20 mg of 2-iminothiolane (SHGel-20) (9) and 40 mg of 2-iminothiolane (SHGel-40) (2) per gram of gelatin. The flow cytometric scatter plots were obtained at 96 h posttransfection using non-cross-linked (C) and cross-linked nanoparticle systems (D). Plasmid DNA-complexed with Lipofectin, a commercially available cationic lipid transfection reagent was used as a control.

cross-linked nanoparticles prepared from thiolated gelatin, namely the SHGel-20, was found to have greater transfection efficiency than the other nanocarriers as well as Lipofectin-pDNA complexes. The results of the study indicate that the thiolation resulted in faster release of their payload in the cells and cross-linking resulted in a particle that remains stable during its transport, and hence these cross-linked, thiolated gelatin nanoparticles could be used for rapid delivery of drugs and genes in vitro. Further, these particles can be used as vectors for systemic delivery of DNA in order to target tissues with a highly reducing environment.

as a function of time as seen in the Figures 10A and 10B. The corresponding scatter plots of the transfected and normal cells at 96 h time point were represented in the form of pixels in regions A1 to A4 in Figures 10C and 10D. The transfected cells were expressed as a percentage of the total cells present in quadrants A3 and A4. Among the non-cross-linked systems the two thiolated gelatins showed better transfection than the gelatin nanoparticles but lower than the Lipofectin-pDNA-treated cells. Upon cross-linking, the SHGel-20 nanoparticles showed better transfection properties than either SHGel-40 or Lipofectin-pDNA. The results can be explained by the fact that cross-linking resulted in a stable particle, which probably provided stability to the payload in the cells by protecting it until the DNA reached the perinuclear region. In the case of SHGel-40 nanoparticles, the polymer would probably be slightly more cytotoxic than the SHGel-20 and showed lesser transfection efficiency due to its effect on cell viability. When incubated for longer periods of time the protein expressed is cytotoxic to the cells resulting in apoptosis or cell death, which led to a decrease in the percentage of transfected cells.

This study was supported by a grant RO1-CA095522 from the National Cancer Institute of the National Institutes of Health. The flow cytometry studies were performed at the Forsyth Dental Institute (Boston, MA). We deeply appreciate the assistance of Dr. Jean Eastcott with the flow cytometric experiments and results interpretation.

CONCLUSIONS

LITERATURE CITED

Gelatin was thiolated successfully using the 2-iminothiolane reagent, and the thiolated biopolymer was used to prepare nanoparticles by desolvation method. A well-characterized nanoparticulate system with a size of about 200 nm and negative surface charge was achieved. The in vitro release studies resulted in a significantly greater release of encapsulated macromolecular payload (FITC-dextran) from the non-cross-linked as compared to the cross-linked nanoparticles both in the presence and absence of GSH. The release from thiolated gelatin nanoparticles was enhanced in the presence of GSHcontaining PBS than PBS alone. Both the non-crosslinked and cross-linked thiolated and control gelatin nanoparticles carrying the EGFP-N1 transgene were internalized by the NIH-3T3 cells, and the expression of the green fluorescent protein was observed as early as 6 h after transfection. Upon quantitative analysis, the

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BC050146T

Preparation and Evaluation of Thiol-Modified Gelatin Nanoparticles for

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