Gelatin is a natural, biocompatible, and nontoxic macromolecule, obtained by the partial hydrolysis of collagen derived from the skin, white connective tissue, and bones of animals. Various types of
gelatin are available based on the preparation method from collagen, the source of collagen, conditions during extraction, pH, thermal history, electrolyte, and impurities or additives. In general, gelatin obtained by an acid‐treated hydrolysis is known as type A, and gelatin obtained by a base‐treated process is known as type B. Gelatin has average molecular weight ranging from 20,000 to 250,000 Da. The protein fractions include various amino acids linked together by an amide linkage, with the major amino acids being glycine, proline, hydroxyproline, and glutamic acid accounting for 25.5%, 18.0%, 14.1%, and 11.4%, respectively [27]. Mainly, the functional groups of the amino acids and the N‐ and C‐terminal groups result in the amphoteric character of gelatin; that is, in acidic solution, the polymer is positively charged, whereas, in alkaline solution, it is negatively charged. However, gelatin possesses an isoelectric point ranging between 4.8 and 9.4, with acid‐
processed gelatin having higher isoelectric points than base‐processed gelatin [28]. Gelatin is an attractive agent owing to the fact that it is natural, biodegradable, biocompatible in physiological environments, edible, water permeable, and insoluble in cold water, whereas completely soluble in hot water. Drawbacks related to its animal origin, such as eventual remnants of animal proteins or infectious agents, may be circumvented using recombinant technology to enhance reproducibility and safety of the material. Gelatin is widely used in pharmaceutical, medical, cosmetic and food products as gelling, thickening, binding, and stabilizing agent [27].
Gelatin is commonly formulated as micro‐ or nanospheres. However, gelatin is usually cationized first by introducing amino groups via the addition of ethylenediamine and ethylene dichloride [29, 30]. In order to produce microspheres, the emulsification‐coacervation method in water‐in‐oil (w/o) emulsions is adopted, where aqueous solution of cationized gelatin is added dropwise into the oil phase under continuous stirring to yield a water‐in‐oil emulsion. After the gelation of the gelatin aqueous solution, the resulting microspheres are collected, washed with ice‐cold acetone and crosslinked with glutaraldehyde [29, 31]. On the other hand, gelatin nanospheres are produced from cationized gelatin by a coacervation method, where acetone or ethanol, as a non‐solvent, is added to aqueous gelatin solution under stirring. Likewise, the resulting nanospheres formed can be cross‐linked by adding glutaraldehyde [30, 32]. Besides cationization, other gelatin‐modifications reported include PEGylation to increase in vivo stability and elongate circulation times [33], thiolation to promote intracellular delivery to rapidly proliferating tumor cells [34] as well as incorporating specific targeting ligands for active cell targeting such as epidermal growth factor receptor (EGFR)‐targeting peptide [35].
Moreover, another formulation strategy was reported by Kadengodlu et al. [36] where they developed cationic cholesterol‐modified gelatin (cCMG) that formed polymeric micelles in an aqueous solution and demonstrated an excellent ability in delivering siRNA in vivo. Cholesterol was modified with an amino group. By the 1‐ethyl‐3‐ (3‐dimethylaminopropyl) carbodiimide (EDC) coupling method, the amino‐modified cholesterol was allowed to conjugate with the carboxyl groups of the Asp and Glu residues present in the recombinant human gelatin (rhG), which ultimately quenched the negative charges. Therefore, when dispersed in an aqueous solution, cCMG displayed an overall positive charge and was found to be cationic in nature. cCMG showed superior stability wherein neither the size nor the zeta potential of the polymeric micelles were affected after incubation at 37oC in PBS for 24 h. The positively charged cCMG formed a stable complex with the negatively charged siRNA and therefore increased the half‐life of siRNA by protecting it from enzymatic degradation.
Moving to in vivo experiments, earlier reports of siRNA delivery utilized cationized gelatin microspheres to deliver plasmid DNA (pDNA) expressing small interference RNA. Matsumoto et al.
[37] complexed cationized gelatin with pDNA coding for anti VEGF siRNA, with the aim to increase cellular uptake and prolong the release of the siRNA following an intratumoral injection. Results showed that a marked reduction in vascularity accompanied the suppression of VEGF production in the transfected tumor. Fluorescent microscopy studies showed that the complex of VEGF siRNA with cationized gelatin microspheres was still present in the tumor 10 days after injection, while free siVEGF had vanished by that time. These data suggest that cationized gelatin microspheres containing VEGF siRNA can be used to normalize tumor vasculature and inhibit tumor growth in a NRS‐1 squamous cell carcinoma xenograft model. A similar approach was adopted by Kushibiki et al. [38] who investigated the feasibility of using cationized gelatin complexed with pDNA encoding for anti‐transforming growth factor‐β (TGF‐β siRNA) for the treatment of renal interstitial fibrosis, a common pathway of chronic renal disease, leading to end‐stage renal failure. TGF‐β is one of the primary mediators that induce accumulation of extracelluar matrix in the fibrotic area, therefore it was expected that local suppression of TGF‐β receptor is one of the crucial strategies for anti‐
fibrotic therapy. A pDNA of TGF‐ β R siRNA expression vector with or without complexation of a cationized gelatin was injected to the left kidney of mice via the ureter. Unilateral ureteral obstruction (UUO) was performed for the injected mice to evaluate the anti‐fibrotic effect. Injection of pDNA‐cationized gelatin complex significantly decreased the level of TGF‐β and α‐smooth muscle
actin over‐expression, the collagen content of mice kidney, and the fibrotic area of renal cortex, in comparison to free pDNA injection.
More recently, Xia et al. [39] utilized cationized gelatin microspheres (CGM) loaded with anti‐Heat Shock Protein 47 siRNA (HSP47 siRNA) in an attempt to reduce the progression of renal tubulointerstitial fibrosis in a mouse model, as HSP47 is a collagen‐specific molecular chaperone and plays an essential role in regulating collagen synthesis. Treatment with the HSP47 siRNA‐CGM complex prolonged the blocking effect for HSP47 expression compared to HSP47 siRNA administered alone, and consequently, the HSP47 expression was markedly diminished even after 14 days and amelioration of the collagen accumulation was observed, indicating a prolonged activity of the delivery system. Successively, the same group went further with this therapeutic system (HSP47 siRNA‐CGM) and reported successful prolonged activity in suppressing the development of peritoneal fibrosis in mice [29]. Authors have shown that release of the siRNA cargo was continued over 21 days due to the slow degradation of gelatin microspheres, and thus, a single injection of (HSP47 siRNA‐CGM) suppressed collagen expression and macrophage infiltration, thereby preventing peritoneal fibrosis. Nakamura et al. [31] also reported about efficient controlled delivery of siRNA molecules from cationized gelatin microspheres. With the aim to treat autoimmune alopecia (alopecia areata), the group has loaded T‐box 21siRNAs conjugated into cationized gelatin microspheres to suppress the expression of the interferon‐γ gene, believed to play a crucial role in this disease, and administered them by subcutaneous injection to alopecic mouse models. Cationized gelatin microspheres were gradually processed in the skin and, in turn, released their cargo of siRNA slowly into the surrounding tissue. Cationized gelatin‐conjugated T‐
box 21 siRNA injections were more effective in improving hair growth than naked T‐box 21 siRNA injections or non‐sense siRNA conjugated with cationized gelatin. There was no recurrence of alopecia in the mice during a 2‐month observation period after the cessation of T‐box 21 siRNA application. This might be attributed to the successful elongated suppression of T‐box 21 protein as proved by Western blotting.
Further developments for oral delivery of siRNA were reported using a multi‐compartmental particle system called NiMOS, which stands for “Nanoparticles‐in‐Microspheres Oral System”. The idea behind is to protect the therapeutic load and facilitate their uptake into GIT tissue cells of interest by prolonging exposure time of the encapsulating particles with the targeted cells, and controlled release of the payload via slow dissolution of the vehicle matrix. The delivery system is
composed of gelatin nanoparticles encapsulated within poly(ε‐caprolactone) microspheres and was proven to have promising potential for oral delivery of pDNA [40]. In an attempt to improve current treatments for inflammatory bowel disease (IBD), Kriegel et al. employed NiMOS to deliver anti‐
TNF‐α siRNA orally [32], and afterwards to deliver both anti‐TNF‐α siRNA and cyclin D1 (CyD1) siRNA in one oral formulation [41]. The central hypothesis of this work was that gene down‐
regulation or silencing of one or more pro‐inflammatory cytokines involved in IBD such as TNF‐α, accomplished by successful oral siRNA delivery, can at least temporarily restore the delicate balance between pro‐ and anti‐inflammatory markers. Experiments aim to evaluate the gene silencing capability of multiple genes through dual oral siRNA delivery as a means to further alleviate the symptoms of IBD in a murine model of colitis, thus intensifying the silencing of pro‐
inflammatory markers and multiplying the efficacy of the treatment. Small interfering RNA targeted against TNF‐α and CyD1 was entrapped within NiMOS either separately or in combination. Blank NiMOS, NiMOS loaded with scramble or inactive siRNA sequence were used as controls. Three days after the last NiMOS dose had been received, combined siRNA treatment of TNF‐α and CyD1 resulted in a more potent down‐regulation of TNF‐α mRNA expression rather than single siRNA administration. At a later time‐point, all silencing NiMOS (TNF‐α, CyD1, TNF‐α/CyD1) resulted in significantly reduced TNF‐α mRNA levels with respect to all remaining control groups, with minor differences in their transcription level. Dual treatment was not able to silence CyD1on an mRNA level as effectively as single treatment of CyD1 NiMOS, which can potentially be ascribed to a dilution effect stemming from the TNF‐α siRNA. In addition, successful gene silencing with the aid of dual siRNA treatment suppressed expression of several pro‐inflammatory cytokines (interleukin‐
1α and ‐β, interferon‐γ), an increase in body weight, and reduced tissue myeloperoxidase activity, while the silencing effect of CyD1 siRNA or dual treatment was more potent than with TNF‐α siRNA alone [41].
5 Cyclodextrins (CDs)
CDs are enzymatically modified starch derivatives with a donut shape or a truncated cone and are made from D‐glucopyranose units connected through α‐(1‐4) linkages. Each CD type is designated by a Greek letter according to the number of glucose units. For instance, α‐, β‐ and γ‐CDs are produced commercially and consist of 6, 7 and 8 glucose units, respectively (Figure 2). All these exhibit low toxicity [42]. The three‐dimensional structure of a CD is such that the inner cavity
surface is hydrophobic, and the exterior of the cavity is hydrophilic, giving a unique structure that enables the CDs to include a wide variety of compounds to form host-guest inclusion complexes.
Among the three natural CDs, the β‐CD is currently the most widely used, and also the most thoroughly studied. CDs have been proven to be safe and tolerable in numerous toxicological and clinical tests and the use of CDs in controlled release applications is well known [43].
Figure 2: (a) Chemical structure of the three main types of cyclodextrins. (b) Truncated cone structure of γ‐
CD.
CDs have recently been applied as delivery vehicles for siRNA, which has led to a surge of interest in this area [44]. However, for complexing negatively charged siRNA molecules, a positive charge is needed. Thus, "Cyclodextrin containing polycations" also known as "Cyclodextrin based polymers"
(CDPs) were used. CDP is a short polycation (Figure 3) in the form of linear polymer, formed by a condensation polymerization reaction between a bi‐functionalized CD co‐monomer and a second bi‐functionalized comonomer that contains the positive charge centers. CDPs assemble with nucleic acids (polyanions) primarily via electrostatic interactions forming nanoparticles where the nucleic acids are completely protected from nuclease degradation [45].
Figure 3: Schematic of linear, water‐soluble cyclodextrin containing polymers (CDPs). (a) Generalized structure of polymers where A represents a segment of the polymer that comes from one comonomer (typically containing the cyclodextrin), B represents a segment of the polymer that comes from the other comonomer, C is the charge center, and S is the spacer between A and C. (b) Specific example of a polymer with labeled A, B, C, and S. “Adapted with permission from ref [108]. Copyright 2004 CRC Press. Taylor and Francis Group”.
CDPs are very interesting structures not only because they maintain CD tolerability and lack of immunogenicity [46], but also because the CD units residing on the surface of the particles are used to functionalize the particle with moieties conferring stability, specific targeting and endosome escaping properties to the dosage form (Figure 4). For example, CD is able to form inclusion complex with adamantane (AD). This property was used to functionalize the CDP‐siRNA particles
with the steric stabilization agent adamantane‐PEG (AD‐PEG) and the targeting agent adamantane‐
transferrin (AD‐PEG‐Tf) for targeting cancer cells. Moreover, to improve the particles’ ability to escape endosomal vesicles, imidazole moieties were incorporated which were reported to cause a pH buffering effect [47]. Alternatively, the presence of amino‐cholesterol derivatives induces conversion of membrane lipids from the Lα to the HII phase, thereby causing disruption of the endosomal membrane under endosomal pH conditions [48]. Both techniques and others enhance siRNA release into the cytoplasm. The fabrication, functionalization and different applications of CDPs have been recently reviewed [44‐46].
Figure 4: Schematic representations of the Adamantane‐PEG (AD‐PEG), Adamantane‐PEG‐transferrin (AD‐
PEG‐Tf) inclusion complex formation with the cyclodextrin (CD) aiming to functionalize a CDP‐siRNA particle.
"Adapted with permission from (Davis, M.E. The first targeted delivery of siRNA in humans via a self‐
assembling, cyclodextrin polymer‐based nanoparticle: from concept to clinic. Mol Pharm, 2009. 6(3): p. 659‐
68.). Copyright (2009) American Chemical Society."
Several authors have published about various designs of CD‐based delivery systems of siRNA, however, according to the scope of this chapter we will describe only selected examples of these trials that moved beyond cellular tests to in vivo studies. Hu‐Lieskovan et al. [49] used a CDP structure similar to the one illustrated in Figure 4, to study the systemic delivery of sequence‐
specific small interfering RNA (siRNA) targeting knockdown of the EWS‐FLI1 gene. The particle included imidazole‐modified CDP to bind and protect siRNA, transferrin as a targeting ligand for delivery to transferrin receptor–expressing tumor cells, and PEG at the particle surface for stabilization. Both, transferrin and PEG were attached via inclusion complex formation between the terminal adamantane and the cyclodextrins. The delivery system efficiently inhibited tumor
growth in a murine model of metastatic Ewing’s sarcoma where only 20% of the mice receiving treatment showed any tumor growth compared with 90 to 100% in other control groups. Removal of the targeting ligand or the use of a control siRNA sequence eliminated the antitumor effects.
Additionally, no abnormalities in IL‐12 and IFN‐α, liver and kidney function tests, complete blood counts, or pathology of major organs were observed during long‐term administrations. The same delivery system was used for an in vivo toxicological study by Heidel et al. [50] to deliver escalating i.v. doses of siRNA targeting the M2 subunit of ribonucleotide reductase to cynomolgus monkeys.
Administration of doses of 3 and 9 mg siRNA/kg, respectively, were well tolerated. At 27 mg siRNA/kg, a very high dose about 20–100 times larger than those that have shown efficacy in mouse models (when doses are extrapolated across species on a body surface area basis), elevated levels of blood urea nitrogen and creatinine are observed that are indicative of kidney toxicity. Mild elevations in alanine amino transferase and aspartate transaminase at this dose level indicated that the liver was also affected to some extent. Also, a mild increase in immune response at high doses was noted.
In another report, Guo et al. [51] designed targeted cyclodextrin‐based nanoparticles loaded with anti‐VEGF siRNA and examined their efficacy in a mouse prostate carcinoma model. Β‐CD was modified on the primary side with a cationic group, guanidino, which is reported to enhance cell penetration [52] beside its role in complexing the siRNA. A click chemistry approach was used to modify the CD on the secondary side with a PEG chain to produce G‐CD‐PEG. The PEG chain was designed to mask any residual charge, to reduce toxicity and to enhance stability. In addition, the PEG chain was used as a spacer to attach the targeting ligand anisamide resulting in the synthesis of a targeted PEGylated CD, G‐CD‐PEG‐AA. Anisamide moiety is reported to have high affinity to the sigma receptor, a membrane‐bound protein known to be overexpressed in many human malignancies including prostate cancer. In vivo studies showed that neither naked VEGF‐siRNA, G‐
CD‐PEG‐AA alone, nor VEGF‐siRNA formulated with G‐CD‐PEG (without the anisamide targeting moiety), had a significant effect on tumour growth compared to the negative control (PBS). Only G‐
CD‐PEG‐AA/VEGF‐siRNA particles achieved a significant (p < 0.05) inhibitory effect relative to the vehicle alone and approximately a 3‐fold reduction in tumor volume relative to PBS, indicating that the improved down‐regulation of VEGF expression is due to site‐specific delivery of siRNA by G‐CD‐
PEG‐AA.
Brahmamdam et al. [53] have reported a potential application of siRNA‐based therapy for the treatment of infectious diseases. The authors used a CDP‐based transferrin‐ targeted delivery vehicle to administer siRNA to Bim and PUMA, the two key cell death proteins that are markedly upregulated during sepsis. The study was conducted in mice immediately after cecal ligation and puncture to show that antiapoptotic therapy could markedly decrease lymphocyte apoptosis and prevent the loss of splenic CD4 T and B cells as confirmed by flow cytometry. The study also demonstrated the possibility of simultaneous targeting of two critical mediators of sepsis‐induced apoptosis. The system effectively delivered the siRNA to immune effector cells with no noticeable off‐target effects, suggesting a major advancement for the treatment of infectious disease.
Grafting CD units with dendrimers has gained increasing interest in the scientific community.
Dendrimers are highly branched tree‐like polymers that can be used in masking the charge of siRNA long enough for in vivo delivery. Among the various dendrimer conjugates with CD units, the dendrimer (G3) conjugate with α‐CD displayed remarkable properties as siRNA delivery carrier [54].
Hayashi et al. [55] prepared lactosylated dendrimer (G3) conjugates with α‐cyclodextrin (Lac‐α‐CD (G3)) as novel hepatocyte‐specific siRNA carriers in order to treat transthyretin (TTR)‐related familial amyloidotic polyneuropathy (FAP). Functionalization with lactose was found to improve asialoglycoprotein receptor (AgpR)‐mediated hepatocyte‐selective nucleic acids transfer activity, both in vitro and in vivo. The Lac‐α‐CD/siRNA complexes at the dose of 1 mg/kg siRNA lowered TTR mRNA expression suggesting that the Lac‐α‐CD/siRNA complex has the potential to induce the in vivo RNAi effect in the liver of mice after intravenous administration. Furthermore, to improve the siRNA transfer activity and to solve the lack of target specificity of α‐CD (G3), Arima et al. [56]
prepared folate‐polyethylene glycol (PEG)‐appended α‐CDs (G3) with various degrees of substitution of folate (DSF), and evaluated their siRNA transfer activity to folate receptor (FR)‐
overexpressing cancer cells in vitro and in vivo. Of the three Fol‐PEG‐αCDs (G3, DSF 2, 4 and 7), Fol‐
PEG‐αCD (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR‐positive). Fol‐PEG‐αCD (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol‐PEG‐αCD (G3, DSF 4) was observed in KB cells (FR‐positive) or A549 cells (FR‐negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol‐PEG‐αCD (G3, DSF 4) elicited neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol‐PαC (G3, DSF 4) tended to show in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells‐bearing mice, and fluorescently labeled Fol‐PEG‐αCD (G3, DSF
4) were shown to accumulate in tumor tissues after intravenous injection in these mice. More information on the use of dendrimers‐CD conjugates can be found in this review [54].
Interestingly, Calando Pharmaceuticals (Pasadena, CA, USA) has been successful in advancing their CD based siRNA/oligonucleotide nanoparticle delivery (RONDEL) technology toward clinical application [45, 46]. This platform consists of a CDP, PEG as a steric stabilizing agent and human Tf as the targeting ligand for binding to Tf receptors that are typically upregulated in cancer cells. The
Interestingly, Calando Pharmaceuticals (Pasadena, CA, USA) has been successful in advancing their CD based siRNA/oligonucleotide nanoparticle delivery (RONDEL) technology toward clinical application [45, 46]. This platform consists of a CDP, PEG as a steric stabilizing agent and human Tf as the targeting ligand for binding to Tf receptors that are typically upregulated in cancer cells. The