Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan–plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT–PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or Mn in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.
Fibroblast growth factor-2 (FGF-2) is a member of the FGF family that currently consists of at least 18 homologous proteins that bind FGF receptors, including the two prototypes, acidic FGF (FGF-1) and basic FGF (FGF-2). Both FGF-1 and FGF-2 lack a classical signal sequence for secretion through the conventional endoplasmic reticulum-Golgi apparatus.1, 2, 3, 4 Several reports indicate that a variety of forms of FGF-2 are produced as a result of N-terminal extensions, which apparently affect localization of FGF-2 in cellular compartments but do not affect biological activity. FGF-2 may play a role in vivo in the modulation of such normal processes as angiogenesis, wound healing and tissue repair, embryonic development and differentiation, and neuronal function and neural degeneration.1, 2 It was reported that chimeric expression of FGF-1 fused with a signal peptide of FGF-4 enhanced the secretion efficiency in transfected NIH3T3 cells.4 To increase the secretion level of FGF-2 in our study, a recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 with eight amino-acid residues of the signal peptide of FGF-4.
Platelet-derived growth factor (PDGF) is a glycosylated, disulfide-linked dimer.5 There are two types of polypeptide, A (16 kDa) and B (14 kDa), with about 50% sequence identity, disulfide linked into three possible dimeric molecules, PDGF-AA, -AB, and -BB. PDGF acts on many cells, especially mesenchymal cells, as a mitogen and a chemotactic factor. PDGF promotes wound healing and may contribute to neoplastic transformation.6, 7 The major source of PDGF in human blood is platelets, in which PDGF-AA and -AB are stored in α-granules and released when platelets are activated. In the absence of platelet activation, there is little PDGF in plasma, and added PDGF is cleared rapidly. PDGF is present at all stages of the wound healing cascade.8, 9 PDGF-BB, the most potent of the three PDGF isoforms,10 is the only growth factor to date to show clinical effectiveness in human chronic wound healing, with few reported adverse events as a topical agent applied to debrided wounds.8, 11 It has been recognized that growth factors contribute to tissue regeneration at various stages of cell proliferation and differentiation. FGF-2 and PDGF-BB are known to be good candidates for enhancing the repair of cartilage lesions or enhancing bone defect fill in a human clinical trial.12, 13 Recent studies have suggested a synergy between PDGF-BB and FGF-2 in producing more competent vascularization.14
In our study, these recombinant FGF-2 and PDGF-BB genes were cloned into the expression vector pVax1− with a cytomegalovirus promoter and delivered into experimental animals through both subcutaneous (SC) and intramuscular (IM) injection routes. It is beneficial that delivered genetic material be protected by a biocompatible, safe, and efficient carrier. Chitosan is such a natural biodegradable cationic polymer that is extracted from crustacean shells and has been studied earlier as a non-toxic gene carrier for in vitro gene transfection.15, 16 As a natural cationic polymer of glucosamine that is partly acetylated, chitosan possesses the advantage of forming polyelectrolyte complexes for DNA delivery that have improved safety over viral systems, ease of synthesis, low cost, no limit of DNA size, and an adjustable structure and chemistry of the polymer (molecular weight (MW) and degree of deacetylation (DDA)) to achieve particular properties. We recently found that by controlling and choosing the MW and DDA of chitosan, we could obtain transfection levels in vitro that are similar to those of commercial phospholipid systems.17 Here, we hypothesize that these same chitosan molecular parameters, MW and DDA, can influence ability of these systems to produce the therapeutic proteins FGF-2 and PDGF-BB and their antibodies in vivo, after injection by SC and IM routes.
For production of antibodies, a suitable delivery vehicle is required that will lead to improved presentation of transgene products to antigen-presenting cells (APCs). Chitosan packaging of DNA can improve cell uptake in part, as it is a bioadhesive material that extends the period of contact between the formulation and the targeted tissues. Also, chitosan has an innate ability to tease open tight junctions between cells, thereby, increasing the uptake of carried agents into these cells and increase membrane permeability.18, 19 Consequently, chitosan has been used successfully for nasal delivery of vaccines.20, 21, 22 Intradermal and intranasal immunization was found to induce a comparable peptide and virus-specific CTL responses.20 Other studies show that chitosan nanoparticles can be used for oral immunization to induce a specific immune response.23, 24 More recently, Ghendon et al.25 found chitosan to act as an adjuvant during immunization, as addition of chitosan to the antigen injected parentally resulted in a 4 or 6–10-fold increase in antibody titres after a single dose or with two doses in the case of IM immunization of mice. Other in vivo studies report appreciable protein expression using chitosan as condensing carrier for plasmid delivery. Chitosans with various MW and DDA have been successfully used for systemic,26 IM,27 intratracheal,28, 29, 30 oral,31 topical,32 and direct intestinal15 administration of plasmid DNA. In most of these studies, reporter genes such as luciferase or EGFP were used.
Despite earlier work examining immunization or protein expression with chitosan–plasmid DNA systems, none has specifically examined the effect of DDA and MW of the chitosan on the attained levels of protein expression versus production of neutralizing antibodies. This particular avenue was pursued in this study as we found very significant influences of these parameters (that is, 1000-fold changes) on in vitro transfection efficiency.17 Our results here show that injection of mice with chitosan–plasmid DNA (pDNA) complexes by an SC route led to enhanced levels of systemically circulating therapeutic proteins and immune responses (neutralizing antibodies) as compared with IM injection. However, the specific response obtained depended intimately on the specific chitosan that was used as a complexing agent for pDNA. Among the three that were tested, 92-10-5, 80-10–10, and 80-80-5 [DDA-number average MW (Mn)-amine-to-phosphate ratio (N:P)], we found the higher DDA chitosans (92%) to increase levels of circulating therapeutic protein and reduce the levels of neutralizing antibodies versus the uncomplexed pDNA without chitosan. We found the opposite to be true for lower DDA chitosans (80%) in which immune responses were stimulated to produce greater levels of neutralizing antibodies and correspondingly decreased levels of therapeutic protein compared with the uncomplexed pDNA without chitosan. These results point to interesting possible applications in which the detailed chemistry of the pDNA carrier can be used to significantly influence the desired biological outcome.
Construction of eukaryotic expression plasmids pVax1-4sFGF and pVax1-PDGF-BB
A highly safe plasmid (pVax1) for genetic immunization in animals was selected to construct vectors expressing recombinant FGF-2 and PDGF-BB proteins. All plasmid elements have been optimized and minimized to comply with FDA guidelines for design of DNA vaccines regarding content and elimination of extraneous materials. The eukaryotic DNA sequences in the plasmid are limited to those required for expression to minimize the possibility of chromosomal integration and kanamycin resistance gene for selection in Escherichia coli minimizes allergic responses in hosts. Human FGF-2 lacks a classical signal sequence for secretion through the conventional endoplasmic reticulum-Golgi apparatus, and a low secretion level of FGF-2 has been a main problem in its application of gene therapy. The efficiency of the FGF-2 secretion is improved by replacing nine amino acids from the amino-terminus of the native FGF-2 with eight amino-acid residues of signal peptide of FGF-4 by PCR-based mutagenesis. It has been reported that the recombinant 4sFGF-2 was secreted at levels four times that of the wild-type FGF-2 in COS-7 cells.4 We therefore isolated, cloned, and sequenced its 465 bp PCR product. The cloning strategy that we adopted is represented in Figure 1a where the recombinant 4sFGF-2 gene was cloned in the eukaryotic pVax-1 vector (Figure 1b) where expression is under the control of a cytomegalovirus promoter. DNA sequence analysis showed an exact match with human FGF-2 gene sequence that encodes for 155 amino acids (Figure 1a). For the recombinant plasmid pVax1-PDGF-BB, the analysis of DNA sequence showed 100% homology to human PDGF-BB gene sequence that encodes 109 amino acids.
Characterization of chitosan/pDNA nanocomplexes
All formulations of pDNA–chitosan nanoparticles were in the range of 40–600 nm, as measured by dynamic light scattering and environmental scanning electron microscopy (Figure 2), with the 80-80-5 chitosan giving the smallest particles for both plasmids (Table 1), consistent with recent work showing that longer chitosans condense plasmid DNA into smaller particles.33 As expected, all formulations resulted in positively charged nanoparticles as shown in Table 1 as there was an excess of chitosan. The pH of the formulations ranged from 4.9 to 5.4 and the osmolality ranged from 17 to 19 mOsm kg-1.
Histological and systemic evaluation
The histological examination of IM and SC injection sites showed the presence of chitosan with no specific abnormalities in the injected tissues. For the IM injection sites, the area of injection revealed a mobilization of inflammatory cells surrounding the chitosan early on at 1 day after injection, decreasing over time until a mostly complete resolution of this acute inflammatory response at 14 days after injection for the low MW chitosans 92-10-5 and 80-10-10 (Figures 3a–f and 4a–f). The higher MW chitosan 80-80-5 displayed the most sustained inflammatory infiltrate at 14 days (Figures 5e and f versus 4e and f and versus 3e and f). The SC inflammatory response displayed a similar dependence on chitosan type (e.g. Figures 3, 4, 5g and h).
No deleterious systemic side effects were elicited by exogenous administration of chitosan/pDNA complexes, as the assessment of a variety of clinical and histological parameters (including mortality, behavioral or physical abnormalities indicating systemic or neurological toxicity34 and necropsy of the major organs as heart, kidney, and liver) showed no gross or microscopic changes between the injected and non-injected groups.
Therapeutic protein and neutralizing antibody production by delivery of pVax1-4sFGF-2 and pVax1-PDGF-BB through IM injections
Animal experiments were repeated once using two different batches of animals and two different lots of chitosan, to ensure that in vivo responses reflected the structure and function of the chitosan carrier in terms of DDA and MW, as well as the functional capacity of the immune system. Recombinant protein accumulation and specific antibody production showed similar profiles in the two repetitions, and as a function of chitosan MW and DDA, reflecting constancy in immune system function and modulation within the 63-day period of the study. In the control group (n=8) that received empty plasmid or phosphate-buffered saline (PBS), animals had no changes in the two markers (accumulation of recombinant proteins and production of antibodies). These results reflect typical responses from a population that is not receiving specific intervention to enhance or to modulate immune system function.
Plasmid-mediated muscle-targeted gene transfer offers the potential of a cost-effective pharmaceutical grade therapy delivered by simple IM injections. Muscle has the ability to take up and express engineered genes, and as it is a post-mitotic tissue, their half-life of expression is prolonged. Although muscle is not regarded as a secretory tissue, in many cases, gene products enter the systemic circulation. Figure 6a shows the production and the accumulation of recombinant 4sFGF-2 associated with IM administration of different formulations of chitosan complexed to pVax1-4sFGF-2 plasmids. Formulations 92-10-5 and 80-10-10 in addition to the uncomplexed plasmid produce recombinant protein from day 60 after administration, whereas gene expression level associated to formulation 80-80-5 was hardly detectable (Figure 6a). The high anti-4sFGF-2 antibodies titer observed from day 30 after administration of chitosan 80-80-5/4sFGF-2 complex (Figure 6b) is consistent with augmented APCs at the injection sites according to histological analyses showing a large influx of neutrophils and macrophages into muscle tissue for this chitosan (Figures 5a–f) compared with the lower MW chitosans (Figures 3a–f and 4a–f). The augmented antibody levels for chitosan 80-80-5 therefore appear to repress the accumulation of the recombinant protein. In contrast, the low production level of anti-FGF-2 antibodies associated with formulations 92-10-5 and 80-10-10 (starting only on day 40 after administration) permits the accumulation of systemically circulating recombinant proteins (Figure 6a). These two formulations appear to have transfected cells through IM injection in a manner that limits immune presentation resulting in a continuous production of recombinant FGF-2 that surpasses the capacity of anti-FGF-2 antibodies to prevent its accumulation (Figure 6b). Only the 92-10-5 formulation produced protein levels exceeding the uncomplexed plasmid (Figure 6a) while only the 80-80-5 chitosan produced neutralizing antibody levels that exceed those induced by the uncomplexed plasmid (Figure 6b), showing the specific modulating ability of chitosan type to direct the in vivo response.
The low production levels of recombinant PDGF-BB associated with IM administration are not carrier specific (Figure 7a) but rather probably due to the capacity of muscle tissue (after DNA immunization) to launch a rapid and effective production of neutralizing anti-PDGF BB antibodies (Figure 7b). This interpretation is supported by the higher protein levels found after SC administration of the same complexes described below (Figure 9a). Effectively, the levels of recombinant PDGF-BB obtained by the IM route are 2–3-fold lower (Figure 7a) than those observed with SC administration (Figure 9a) in accordance with the appearance of specific antibodies to PDGF-BB earlier (Day 20) and up to 2-fold higher with IM injection (Figure 7b) compared with SC injection (Figure 9b). The increased antibody levels seen in IM versus SC argue against the higher dose (10 × ) in SC being responsible for these tissue-specific effects. These findings further suggest that recombinant proteins have been immediately neutralized by antibodies already present as confirmed by the results seen in Figure 7b showing antibodies detectable from day 20 after injection that reach maximum levels from day 35 after injection for all chitosan–pDNA complexes, in contrast to the naked plasmid which only produced antibodies after 40 days. In particular, the group of mice injected with chitosan 80-80-5/pVax-PDGF-BB intramuscularly did not show any circulating PDGF-BB, suggesting that this formulation 80-80-5 might be directly processed by APCs that also process the recombinant protein leading to a rapid production of neutralizing antibodies. This interpretation is supported by the inflammatory character of chitosan 80-80-10 seen histologically (Figure 5).
Therapeutic protein and neutralizing antibody production by delivery of pVax1-4sFGF-2 and pVax1-PDGF-BB through SC injection
The ability of biodegradable polymers to facilitate uptake and expression of nanoparticle-associated DNA plasmid by APCs has attracted attention18, 19, 35, 36 and work in our laboratories17, 37 presented here and found in preliminary preclinical data from another study (data not shown) continues to yield promising results for polymer-based formulations. The SC route is proving to be effective for delivery systems using chitosan–DNA complexes. In this study, we found the maximum concentration of recombinant 4sFGF-2 protein in the serum was detected from day 49 after injection with concentration in the range 2300–2600 pg ml−1 of serum (Figure 8a). Moreover, the formulation 92-10-5 induced production of 4sFGF-2 10 days earlier and at levels about 2-fold higher than that of naked pVax1-4sFGF-2. The 4sFGF-2/chitosan 92-10-5 was therefore the most efficient delivery vehicle with transfection levels indicated by systemically circulating recombinant protein that were higher than the plasmid alone and much higher than the other two chitosan–pDNA formulations using 80-10-10 or 80-80-5 in which 4sFGF-2 was hardly detectable (Figure 8a). The fact that protein levels saturated so quickly without any significant antibody production for the 92-10-5 chitosan suggested that cell uptake or processing of the nanoparticles at the injection site may be rate-limiting steps in vivo. In contrast, the chitosan 80-10-10 had the highest antibody titer observed from day 49 after injection (Figure 8b), suggesting that chitosan 80-10-10 can deliver the DNA early enough to produce protein and antibody simultaneously thereby preventing the accumulation of recombinant 4sFGF-2 protein in the serum. Here again these results concur with those described above for IM administration in which the chitosan 92-10-5 is the most effective delivery agent for the therapeutic protein by increasing protein levels and reducing antibody levels compared with the uncomplexed plasmid, while a lower DDA chitosan (80-10-10 in this case) generates an antibody response that abrogates the accumulation of systemically circulating protein (Figures 8a and b).
The levels of recombinant PDGF-BB detected in serum of mice injected with chitosan 92-10-5/pVax-PDGF-BB subcutaneously increased from day 35 after immunization. The group of mice injected with chitosan 80-10-10/pVax-PDGF-BB subcutaneously also show an increasing level of PDGF-BB production but detected only from day 49 after immunization similar to the uncomplexed plasmid (Figure 9a). These results are concordant with the increasing level of anti-PDGF-BB antibodies from day 49 after immunization (Figure 9b). These results may be explained by the fact that high levels of anti-PDGF-BB antibodies may neutralize the recombinant protein at an early stage of its production (mediated by chitosan 80-80-5) and/or inhibit its accumulation (in the case of chitosan 92-10-5 and 80-10-10) (Figures 9a and b). The recombinant PDGF-BB protein levels observed in Figure 9a in the group of mice injected with the three different formulations of chitosan are consistent with high levels of repressive anti-PDGF-BB antibodies in serum from day 49 after immunization as compared with the control group of mice immunized with plasmid alone (empty plasmid, data not shown) or with PBS solution (Figure 9b).
Immunohistochemistry: specific in situ detection of FGF-2 and PDGF
In situ detection of FGF-2 and PDGF-BB confirmed that the nanocomplexes were efficiently taken up by cells, and DNA expression could be achieved depending on the chitosan formulation used and also depending on the plasmid DNA nature [pVax1-4sFGF-2 (data not shown) or pVax1-PDGF-BB] (Figure 10). In situ detection of higher levels of PDGF-BB when delivered with chitosan 92-10-5, compared with low levels obtained with 80-80-5 (Figures 10a–d) agrees with the higher level of production of the recombinant protein measured in the plasma for chitosan 92-10-5.
Gene therapy using non-viral carriers has several potential advantages over the direct use of recombinant proteins, with the most prominent being the ability to achieve sustained high concentrations of growth factors at the targeted site without producing toxic systemic effects.38 In this context, the efficient delivery of growth-promoting genes such as FGF-2 and PDGF-BB locally in a sustained manner is an important goal for effective tissue regeneration, for example, in the treatment of peripheral vascular disease or in the repair of connective tissues such as cartilage and bone. One critical issue for gene therapy in general is the duration of gene expression. For some applications (for example, prevention of ischemia-reperfusion injury after transplantation, immunization for tumor immunotherapy), only transient gene expression is required, and in principle can be achieved using non-viral delivery systems. In our study, we have applied the polymeric gene carrier chitosan, a cationic polysaccharide known for an absence of immunogenicity, high safety, ease of preparation, with no gene sequence limitations and a potential to be used repeatedly.39, 40 Specifically, we examined the in vivo transfection efficiency of the chitosan–pDNA nanoparticles and the expression/distribution of recombinant proteins and their neutralizing antibodies after administration of nanocomplexes through one of two different routes of administration: SC and IM. Earlier studies have shown that plasmid DNA immunization using polymer microspheres have improved potency and excellent promise for therapeutic vaccines41, 42, 43, 44 compared with naked DNA. Unlike naked DNA delivery, nanoparticle-mediated delivery of pDNA can result in efficient, direct delivery to APCs without the need for crosspriming.45
Initially, we successfully constructed recombinant vectors expressing 4sFGF-2 and PDGF-BB proteins using the highly safe vector pVax1 resulting in pVax1-4sFGF-2 and pVax1-PDGF-BB. One means by which one can influence the production of a therapeutic molecule versus the induction of protective immune responses to DNA delivery is by selecting the route of administration. Chow et al.46 have shown that Th1- and Th2-type immunity are dependent on administration route, dose, and the method of injection. This study reveals the effect of two different routes of administration, IM and SC, on recombinant protein expression level and the type of immune response. Our IM injections induced a more potent humoral response than SC administration as manifested by earlier and stronger antibody titers than SC (Figures 5b and 6b versus 7b and 8b). In concordance with this finding, SC administration was associated with greater and more sustained production and accumulation of recombinant protein than for IM administration (Figures 5a and 6a versus 7a and 8a). Both routes of administration produce Th2-type response but act in different manners in terms of raising specific antibodies.35, 36, 47 As the prevalence of APCs is different in muscle than SC tissues, it is likely that the network of APCs residing in the target tissue act as a decision-maker of the extent of humoral response versus accumulation of recombinant protein elicited by these two administration routes for chitosan–pDNA expressing vectors. Furthermore, the differences in the amplitude and in the quality of the immune responses suggest that the APCs transfected in IM and SC locations are functionally distinct and therefore prime the immune response uniquely.48, 49 We have shown that muscular and SC delivery provide a long-term presence of chitosan/DNA expressing vectors and prolongs transgene expression with a specific production of antibodies and recombinant proteins from day 14 to day 63 after administration, in general agreement with earlier studies using other delivery methods.50, 51, 52
A primary outcome of this study arose from direct comparison of chitosans of different MW and DDA as delivery agents for plasmid DNA administered as polyelectrolyte nanocomplexes through both SC and IM routes of administration. Although earlier work in the chitosan field has shown that DDA influences enzymatic degradation of chitosan in which lower DDAs are more degradable,53 additional studies examining immune modulating responses to chitosan have provided variable results, possibly due to different chitosan types (MW and DDA) and preparation methods being used in these studies.54, 55, 56 It is, therefore, critical in any study examining the use of this polymer in a biological environment to accurately account for the influence of chitosan MW and DDA using purified polymer preparations. In this study, we found that chitosan formulations 92-10-5 and 80-10-10 (DDA-Mn-N:P ratio) had the highest in vivo transfection activity in terms of levels of circulating recombinant protein and the 80-80-5 formulation had the lowest protein levels, in close agreement with our earlier in vitro findings.17 Combined with our observation of generally increased protein concentrations when using the SC route of administration as compared with the IM route, these two low MW chitosan formulations are therefore desirable non-viral vectors for protein delivery applications in gene therapy using the plasmids pVax1-4sFGF-2 and pVax1-PDGF-BB through SC administration. The 92-10-5 formulation was particularly interesting given its ability to suppress neutralizing antibody production relative to the plasmid alone. In the case where antibody production is desired for DNA-based vaccines, then the lower DDA formulations such as 80-80-5 appear particularly effective in generating antibody levels several fold higher than the uncomplexed pVax plasmids alone.53, 54, 55, 56
Here, we have shown that IM and SC administration of chitosan/plasmid DNA lead to the expression and distribution of FGF-2 and PDGF-BB recombinant proteins in surrounding tissues, and eventually appeared in serum. Additionally, the recombinant proteins were still detectable at the injection site and surrounding tissues several weeks after administration. This implies that the chitosan/plasmid DNA nanocomplexes were effectively captured by tissues and cells rather than being broken down rapidly.57, 58 Together, these results indicate that specific chitosan–pDNA nanoparticle formulations have great potential as gene carriers for growth factor therapy (for example, 92-10-5) when combined with the recombinant pVax1-4sFGF-2 and pVax1-PDGF-bb plasmids. Furthermore, alternative formulations using lower DDA chitosan (80-80-5) show promise as carriers in DNA vaccines. Overall, the production of recombinant protein was significantly higher after SC administration than after IM administration, indicating that gene delivery through the SC route is an efficient approach. In contrast, in this same context of chitosan formulations used, the production of specific antibodies was rapid and higher when administrated intramuscularly and is promising for the development of prophylactic and/or therapeutic vaccines. This study provides an efficient experimental basis for further studies on safe and efficient gene therapy with FGF-2 and PDGF-bb molecules. It is also worth noting that the therapeutic molecules administered through SC and IM injections in our study can also be administered intravenously or orally and thereby targeted toward novel therapeutics in the areas of regenerative medicine as well as metabolic and infectious diseases.
Materials and methods
The plasmid pVax1 (Cat #V260-20), pCR 2.1 from TA cloning Kit (Cat #45-0046), competent INV α F' cells (Cat # 44-0007), sterile Tris–EDTA (TE) buffer solution pH 7.4 (Cat #60191) and the PureLink HiPure Plasmid DNA purification kit (Cat # K2100-14) were from Invitrogen. Human recombinant PDGF-BB protein (Cat #PHG0044) was from Biosource International. Recombinant human fibroblast growth factor (rhFGF) basic (Cat #234-FSE/CF) at 0.799 mg ml−1 in 10 mM sodium phosphate and 0.3 M glycerin (pH 7.0), the human FGF basic Quantikine ELISA kit (Cat #DFB50), the human PDGF-BB Quantikine ELISA kit (Cat #DBB00) were from R&D Systems, Minneapolis, MN, USA. Sterile Nunc-Immuno 96 MicroWell plates with flat bottom and lid were from Nalge Nunc International, Rochester, NY, USA.
Plasmid construct: pVax1-4sFGF-2
The full-length FGF-2 was generated by RT–PCR. Briefly, the total RNA from human head and neck tumor was extracted by using TRIzol reagent (GIBCO/BRL Life Technologies, Montreal, Canada). A quantity of 5 μg of the purified total RNA was then used to synthesize a cDNA using SuperScripttm One-Step RT–PCR with Platinum Taq kit (Invitrogen, Burlington, Ontario, Canada). For the construction of recombinant 4sFGF-2 plasmid, the amino-terminus of native FGF-2 (amino acids Met1 to Leu9) was replaced with eight amino-acid residues of the signal peptide of FGF-4 (amino acids Met1 to Ala8) by PCR-based mutagenesis. The recombinant 4sFGF-2 was constructed by two separate PCR reactions with two different sets of primers containing nucleotide sequences of the signal peptide of FGF-4 using a set of primers: Fw4s.1 (5′-IndexTermGGGACGGCCGCGCCCGCCTTGCCCGAGGATGGCGGC-3′), Fw4s.2 (5′-IndexTermTGTCGGGGCCCGGGACGGCCGCGCCCGCCTTGCCCGA-3′), and Rv18h (5′-IndexTermCCTCGAGTCAGCTCTTAGCAGACAT-3′). The amplification protocol using Fw4s.1 and Rv18h primers consisted of 35 cycles for 15 s at 94 °C, 25 s at 55 °C, and 25 s at 70 °C. This PCR product was cloned into the EcoRI site of the pCR−2.1 vector. A first round PCR was then performed on this new recombinant DNA plasmid using Fw4s.2 and Rv18h primers followed by a second round PCR using another set of primers [Fw4sFGF (5′-IndexTermGAAGCTTACCATGTCGGGGCCCGGGA-3′) and Rv18h] to amplify the 4sFGF-2 amplicon that was cloned in pCR-2.1 vector. PCR reactions consisted of 35 cycles for 15 s at 94 °C, 25 s at 53 °C, and 25 s at 70 °C. For the expression of the recombinant 4sFGF-2 protein, the insert (465 bp) of pCR2.1-4sFGF-2 plasmid was digested with HindIII and XhOI enzymes and the insert (465 bp) was subcloned into the eukaryotic expression vector pVax-1 (Invitrogen) to obtain the pVax1-4sFGF-2 recombinant plasmid. The 4sFGF-2 sequence was confirmed by Dye-dideoxy sequencing (Roche/Applied Biosystems, Laval, Canada) using universal and specific internal primers in both directions. (Sequencing was performed at McGill University and Genome Québec Innovation Centre Sequencing Platform).
Plasmid construct: pVax1-PDGF-bb
Total RNA was extracted from a healthy blood donor-derived platelet using the TRIzol reagent (GIBCO/BRL Life Technologies) and 5 μg of the purified total RNA served as template to synthesize PDGF-BB cDNA using the SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen, Burlington, Ontario, Canada). The recombinant PDGF-BB was constructed by two separated PCR reactions with the two different sets of primers. The first amplification reaction using the primers FwprePDGF (5′-IndexTermAAGCTTACCATGAATCGCTGCTGGGCG-3′) and RvprePDGF (5′-IndexTermCTCGAGCTAGGCTCCAAGGGTCTCCTT-3′) consisted of 35 cycles for 15 s at 94 °C, 20 s at 57 °C, and 30 s at 70 °C. This PDFG-BB amplicon was submitted to 35 cycles of a nested PCR (15 s at 94 °C, 25 s at 52 °C, and 25 s at 70 °C) to amplify the internal region of the PDGF precursor using the primers FwPDGF (5′-IndexTermAAGCTTACCATGAGCCTGGGTTCCCTGACC-3′) and RvPDGF (5′IndexTerm-CTCGAGGGT CACAGGCCGTGCAGCTGC-3′). The PDGF-BB fragment of 327 bp was then cloned in pCR-2.1 vector. After selection and characterization of positive clones, plasmid DNA was purified and digested with HindIII and XhOI enzymes. The PDGF-BB insert was subcloned into the eukaryotic expression vector pVax-1 (Invitrogen) to obtain the pVax1-PDGF-BB recombinant plasmid. The PDGF-BB sequence was confirmed by Dye-dideoxy sequencing (Roche/Applied Biosystems).
Purification of recombinant plasmids: pVax1-4sFGF-2 and pVax1-PDGF-BB
The plasmids pVax1-4sFGF-2 of 3465 bp and pVax1-PDGF-BB of 3327 bp encode for the human recombinant proteins 4sFGF-2 and PDGF-BB, respectively, each driven by a human cytomegalovirus promoter. The recombinant plasmids were amplified in INV α F' bacteria and purified using Endotoxin-Free Plasmid Maxiprep Kit (Invitrogen). The purified pDNA was dissolved in endotoxin-free Tris–EDTA (TE) and concentration/purity determined on UV spectrophotometer by measuring absorbance at 260/280 nm.
Preparation and characterization of chitosans
Ultrapure chitosan samples (Ultrasan) were provided by BioSyntech Inc. (Laval Qc., Canada) in which quality controlled manufacturing processes eliminate contaminants including proteins, bacterial endotoxins, toxic metals, inorganics, and other impurities. All chitosans had <500 EU g-1 of bacterial endotoxins. Chitosans were selected to have a range of DDA from 98 to 72%. Chitosans of different DDA were depolymerized using nitrous acid to achieve specific number-average MW targets (Mn) of 80 and 10 kDa. For depolymerization, chitosans were dissolved overnight at 0.5% (w/v) in 50 mM hydrochloric acid under magnetic stirring and then treated for 16 h at room temperature with specific amounts of sodium nitrite in the range of 0.001–0.1 mole/mole of chitosan glucosamine. The reaction was stopped by precipitation using 6 N sodium hydroxide to bring the pH above 10. Chitosans were then washed by repeated centrifugation (4000 g for 2 min) and resuspended in deionized water, until the supernatant reached neutral pH. The samples were freeze-dried before characterization and use in the production of chitosan–pDNA nanoparticles. Number- and weight-average MWs (Mn and Mw) of chitosans were determined by gel permeation chromatography as described earlier59 and are reported in Table 2. The DDA was determined by 1H NMR according to Lavertu et al.60
Preparation of chitosan/pDNA nanoparticles
Chitosans were dissolved overnight on a rotary mixer at 0.5% (w/v) in hydrochloric acid using a chitosan glucosamine: HCl ratio of 1:1. Chitosan solutions were then diluted with deionized water to reach the desired amine (deacetylated groups) to phosphate ratio when 1 volume of chitosan would be mixed with 1 volume of pDNA, the latter always at a concentration of 330 mg ml−1 in endotoxin-free TE (10 mM Tris–HCl, 1 mM EDTA, pH 7.4). Before mixing with pDNA, the diluted chitosan solutions were sterile filtered with a 0.2-μm syringe filter. Chitosan/pDNA nanoparticles were then prepared by adding 250 μl of the sterile diluted chitosan solution to 250 μl of pDNA (330 mg ml−1) at room temperature, pipetting up and down and tapping the tubes gently. The resulting chitosan/pDNA nanoparticles solution was then characterized or administered to mice 30 min after preparation without addition of any buffer.
Complex size and zeta potential
Size of chitosan/pDNA complexes was determined by dynamic light scattering at an angle of 173° at 25 °C, using a Malvern Zetasizer Nano ZS (Malvern, Worcestershire, UK). Samples were measured in triplicates using the refractive index and viscosity of pure water in calculations. The zeta potential was measured in duplicate with laser Doppler velocimetry at 25 °C on the same instrument, and with the viscosity and dielectric constant of pure water used for calculations. For both of the above measurements, nanoparticles were diluted 1:10 in water before reading.
High vacuum scanning electron microscopy
An environmental scanning electron microscope (ESEM, Quanta 200 FEG, FEI Company Hillsboro, OR, USA) was used to image nanoparticles to obtain shape and size after complexation with plasmid DNA.
All animal experiments were approved by Institutional Animal Care & Use Committee (University of Quebec). Eighty-four female mice, 4- to 6-weeks old, (Balb/C; Charles River, Canada, CA), each weighing 15–20 g, were used in this study. Chitosan–pDNA nanoparticles were administered to mice either by SC injection on the back (1 ml of total volume; dose of 165 μg of DNA) or by IM injection in both hind legs (50 μl of total volume per leg, total dose of 16.5 μg of DNA). The animals were divided into eight groups of four, according to whether injection was performed subcutaneously (n=32), or intramuscularly (n=32) and eight other mice that received PBS or plasmid alone (empty plasmid) treatment subcutaneously (n=4) or intramuscularly (n=4) were included as negative controls. For histopathology examination, 12 mice were killed at 1, 3, and 14 days after a single SC or IM injection of chitosan/pDNA nanoparticles.
In vivo gene transfection
All experiments were replicated at two different times using two different batches of animals and two different lots of chitosan for the preparation of nanocomplexes. Mice were 5- to 7-weeks old at the first injection. They received seven doses of the nanoparticles in 1- or 2-week intervals on days 0, 7, 14, 21, 35, 49, and 63. Briefly, 1 ml of nanocomplexes was administered subcutaneously through 25G syringe needle (165 μg of pDNA). For IM administration, 50 μl of complexes was injected in each hind leg using a 26G-1/2′′ syringe (8.25 μg of pDNA per leg). The control groups received naked DNA pVax1-4sFGF-2 alone or pVax1-PDGF-BB alone (165 μg subcutaneously and 8.25 μg/leg intramuscularly). Mice that received PBS or plasmid DNA alone, subcutaneously or intramuscularly, were included as negative controls. Every administration was preceded by blood collection from the saphenous vein using a 25G syringe to analyse sera. Animals were killed on day 77.
Histological and systemic evaluation
The group of animals that received single injections (n=12) were killed 1, 3, or 14 days after SC or IM injection of chitosan/pDNA nanoparticules using CO2 euthanasia chambers. The back skin of SC-injected mice was cut with a scalpel and stitched onto a rigid surface. The whole posterior paw of IM-injected mice was cut with a sharpened razor blade at the hip. The skin and the leg were fixed in 10% NBF (Fisher Scientific, Québec, Canada) at room temperature for 1 week. Tissues were trimmed to keep only the quadriceps muscle groups of the thigh and the skin corresponding to the area of the injection site. Samples were alcohol-dehydrated and embedded in paraffin. Sections of 4–6 μm thickness were prepared on a Leica RM 2155 microtome (Leica Microsystems, Deerfield, IL, USA) and collected on microscope slides. Paraffin sections were deparaffinized and rehydrated before staining.
For safranin-O/fast-green/iron-hematoxylin staining, the sections were sequentially immersed in Weigert iron hematoxylin (Sigma Oakville, Canada), 0.04% (w/v) fast green (Sigma) and 1% (w/v) safranin-O (Sigma) in water. For hematoxylin/eosin staining, sections were sequentially immersed in Harris modified Hematoxylin (mercury free; Fisher Scientific, Québec, Canada), 1% LiCO3 (Fisher Scientific) and 0.25% Eosin (Surgypath, Winnipeg, Manitoba) in 80% ethanol and 0.5% acetic acid glacial (JT Baker, Anachemia, Montreal, Canada). Digital images of stained sections were acquired with a Zeiss Axiolab microscope equipped with a Hitachi HV-F22 analog camera. Software for histology analyses included Northern Eclipse (Mississauga, Ontario, Canada).
Quantitative determination of human FGF-2 concentration in sera
The level of FGF-2 production in the sera of mice was determined by ELISA using the human FGF basic Quantikine ELISA kit according to the manufacturer's instructions (Quantikine Human FGF, R&D Systems, Minneapolis, MN, USA). This assay uses the quantitative sandwich enzyme immunoassay. A monoclonal antibody specific for FGF basic was pre-coated onto a microplate. Standards and sera of mice were pipetted into the wells and any FGF-2 present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for FGF-2 was added to the wells. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of FGF-2 bound in the initial step. The color development was stopped, and the intensity of the color was measured at 450 nm, with the correction wavelength set at 570 nm. The optical densities were determined by using a microtiter plate reader (DigiScan Microplate Reader, Phoenix Bio-Tech, Mississauga, canada).
All analyses and calibrations were performed in duplicate. The calibrations on each microtiter plate included recombinant human FGF-2 standards. The blank was subtracted from the duplicate readings for each standard and sample. A standard curve was created using DigiWin software v3.1 from ASYS Hitech GmbH, by plotting the logarithm of the mean absorbance of each standard versus the logarithm of the standard concentration. Concentrations are reported as picograms per millilitre. As samples have been diluted, the concentration read from the standard curve has been multiplied by the dilution factor. The lower detection limit of human FGF-2 was 3 pg ml-1.
Quantitative determination of human recombinant PDGF-BB concentration in sera
Recombinant PDGF protein was determined by ELISA specific for human PDGF-BB. Briefly, a 96-well polystyrene microplate coated with recombinant human PDGF Rß/Fc chimera (Quantikine Human PDGF-BB, R&D Systems, Minneapolis, MN, USA) was used. Standards and sera of mice were pipetted into the wells and any PDGF-BB present was bound by the immobilized receptor. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for PDGF-BB was added to the wells. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of PDGF-BB bound in the initial step. The color development was stopped and the intensity of the color measured at 450 nm, with the correction wavelength set at 570 nm. Results analyses were performed as described earlier with the quantitative determination of human FGF-2 concentrations. The limit of detection of PDGF-BB was 15 pg ml−1.
ELISA assays for antibodies anti-FGF-2 and anti-PDGF-BB detection in sera
The rhFGF basic and the human recombinant PDGF-BB proteins were incubated at a concentration of 100 and 2000 pg well−1, respectively, in a flat-bottom 96-well plate at 4 °C for 12 h. Wells were then washed with a buffer solution (10 mM PBS, 0.05% Tween 20, pH 7.2) and blocked with PBS, containing 2% bovine serum albumin, 0.1% Tween 20 for 90 min at room temperature. After a second step of washing, serum samples were diluted (1:200) in PBS, containing 1% bovine serum albumin, pipetted into the wells and incubated at room temperature for 1 h. The wells were washed five times, affinity purified biotinylated goat anti-mouse immunoglobulins diluted in PBS (1:1500) containing 1% bovine serum albumin (Sigma) were added for 1 h at room temperature. After a further wash, extravidin-peroxidase was diluted in PBS (1:500) containing 1% bovine serum albumin, added to the wells and incubated 30 min at room temperature. Finally, visualization of positive interaction was confirmed through the addition of 100 μl of 3,3′,5,5′-tetramethyl-benzidine (Sigma) and incubation in the dark for 20–30 min at room temperature. The reaction was stopped using stop reagent 3,3′,5,5′-tetramethyl-benzidine substrate for ELISA (Sigma) and the assay results were measured at 450 nm (reference filter was at 570 nm) using the DigiScan Microplate Reader and the DigiWin Software v3.1 from ASYS Hitech GmbH.
Immunohistochemistry: specific in situ detection of FGF-2 and PDGF
The group of animals that received sequential injections (n=72) were killed using CO2 euthanasia chambers. An indirect immunoperoxidase assay was used to examine FGF-2 and PDGF-BB protein expression and distribution in formalin-fixed, paraffin-embedded tissue sections (4–6 μm thickness). Affinity-purified anti-FGF-2 polyclonal antibodies raised against a synthetic peptide corresponding to amino acids 40–63 of human FGF-2 were obtained from Oncogene Research Products (Cambridge, MA, USA). The purified rabbit polyclonal antibody that recognize the B form of human PDGF was raised against a synthetic peptide corresponding amino acids 101–116 of human PDGF B chain. All incubations were performed at room temperature (20 °C) in a humidified chamber unless otherwise stated. Briefly, the sections were deparaffinized in three changes of xylene and hydrated through a series of ethanol solutions of graded concentration. Intrinsic endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide/20% methanol. Sections were incubated 2 h with primary antibodies against FGF-2 at 1:100 dilution and PDGF-BB at 1:200 dilution in a humidity chamber. Subsequent steps were done using Calbiochem (VWR Canlab, Mississauga, Canada, Immunostaining Biotin/Streptavidin kit, anti-Rabbit IgG, Peroxidase) and ZYMED Laboratories (Invitrogen, Burlington, Canada, Liquid 3,3′-diaminobenzidine plus substrate) kits according to the manufacturer's protocols. After counterstaining with hematoxylin, sections were dehydrated and coverslipped with Permount (Fisher). For negative controls, in each run, some sections were processed after omitting the primary antibody or by substituting normal goat serum.
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This work was supported by the Canadian Institutes of Health Research (CIHR). The authors thank Liviu Dragomir, Viorica Lascau, Genevieve Picard for assistance with histological preparations and Dr Monica Nelea for ESEM analysis.
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Jean, M., Smaoui, F., Lavertu, M. et al. Chitosan–plasmid nanoparticle formulations for IM and SC delivery of recombinant FGF-2 and PDGF-BB or generation of antibodies. Gene Ther 16, 1097–1110 (2009). https://doi.org/10.1038/gt.2009.60
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