In Situ Gel Formulations for Gene Delivery: Release and Myotoxicity Studies

The in vitro release of plasmid DNA and salmon sperm DNA from in situ gel formulations was investigated. Two in situ gel systems were studied: (a) an interpolymeric complex (IPC) of water-soluble polymers polymethacrylic acid (PMA) and polyethylene glycol (PEG) and (b) a hydroxypropylmethylcellulose–carbopol system (H:C). Two-way analysis of variance with replication demonstrated that both gel composition and medium pH influenced significantly the release of plasmid DNA from in situ gel formulations. When the release of both types of DNA was compared, higher release was observed for plasmid DNA compared to genomic salmon sperm DNA. Conformational analysis of the released plasmid DNA showed that DNA was released without degradation, but with remarkable conversion from supercoiled (SC) to open circular (OC). In addition, the tested in situ gel systems demonstrated protection from DNAse I degradation. The myotoxicity of the injectable gelling solutions was assessed by the cumulative release of creatine kinase (CK) over 120 min from the isolated rodent extensor digitorum longus (EDL) muscle. A higher level of cumulative CK was observed for IPC when compared to H:C (2:1). These results demonstrate that the in situ gelling systems can be considered as a valuable injectable controlled-delivery system for pDNA in their role to provide protection from DNAse degradation.     

Novel Nasal in situ Gelling System for Treatment of Sinusitis

In situ gelling systems based on temperature-dependent phase transition containing pheniramine and phenylephrine were developed using combination of Poloxamers, different cellulose polymers (HPMC) and xanthan gum. The formulations were tested for in vitro gelation, appearance, pH, drug content, in vitro drug release, mucoadhesive strength, preservative efficacy and stability. In vitro release studies revealed significant prolonged released of both drugs up to 24 h as against only 2 h with drug solution. Formulations were found to stable over period of 3 months. In vivo nasal residence time of in situ gel by gamma scintigraphy was found to be significantly higher (6 h) in comparison to drug solution (15 min). It can be concluded that Poloxamers in combination with HPMC are suitable to develop stable, safe in situ temperature-based mucoadhesive gelling systems with prolonged nasal residence time.     

Characterization of thermosensitive chitosan-based hydrogels by rheology and electron paramagnetic resonance spectroscopy.

Chitosan, an amino-polysaccharide, has been proposed as a promising biopolymer for tissue repair and drug delivery. Chitosan solutions containing glycerol-2-phosphate (beta-GP) have been described as injectable in situ gelling thermosensitive formulations, which undergo sol-gel transition at physiological pH and temperatures. This feature makes them suitable for the parenteral administration of drugs, especially for peptides and proteins. The aim of the present study was to get a deeper insight into the macro- and microstructure of chitosan/beta-GP systems. In addition to oscillating rheology, electron paramagnetic resonance (EPR) spectroscopy was applied to examine the microviscosity and pH inside the gels depending on the beta-GP concentration and to follow the loading and release of spin-labelled Insulin. All chitosan/beta-GP solutions showed a physiological pH ranging from 6.6 to 6.8 that did not change during gelation, irrespective of the proportion of beta-GP. The dynamics of the spin-labelled Insulin and its microviscosity inside the gels and during release were monitored by EPR spectroscopy. The results indicate that the Insulin was incorporated into the aqueous environment of the gel and was released in its native form. The in vitro drug release from the gels was governed by diffusion of drug from the gel matrix. A sustained release of Insulin was observed over a period of 2 weeks. Increasing the proportion of beta-GP increased the amount of released Insulin and the velocity thereof.     

Development and characterization of thermosensitive pluronic-based metronidazole in situ gelling formulations for vaginal application

The purpose of this study was to develop pluronic-based in situ gelling formulations of metronidazole (MTZ) for treatment of bacterial vaginosis, aimed at prolonging the residence time, controlling drug release, enhancing efficacy, decreasing recurrence, and increasing patient compliance. The in situ gel formulations were prepared using different concentrations of pluronic F-127 (PF-127) alone and in combination with pluronic F-68 (PF-68). The prepared formulations were evaluated for their gelation temperature (T(gel)), in vitro drug release, rheological properties, mucoadhesion properties and tolerability by vaginal mucosa in tissue levels. The T(gel) decreased with increasing PF-127 concentration. The T(gel) was modulated by addition of PF-68 to be within the acceptable range of 25-37 °C. With increasing pluronic concentration, the in vitro drug release decreased, viscosity and mucoadhesive force increased. Histopathological examination of rabbit vaginas from the control and treated groups revealed normal histology of the vagina and cervix. Based on the in vitro evaluation of prepared formulations, the in situ gelling liquid formulated with PF-127/PF-68 (20/10 %, m/m) was selected for further clinical evaluation.     

Development of thermosensitive chitosan/glicerophospate injectable in situ gelling solutions for potential application in intraoperative fluorescence imaging and local therapy of hepatocellular carcinoma: a preliminary study

Objectives: Thermosensitive chitosan/glycerophosphate (C/GP) solutions exhibiting sol–gel transition around body temperature were prepared to develop a class of injectable hydrogel platforms for the imaging and loco-regional treatment of hepatocellular carcinoma (HCC). Indocyanine green (ICG) was loaded in the thermosensitive solutions in order to assess their potential for the detection of tumor nodules by fluorescence.

Methods: The gel formation of these formulations as well as their gelling time, injectability, compactness and resistance of gel structure, gelling temperature, storage conditions, biodegradability, and in vitro dye release behavior were investigated. Ex vivo studies were carried out for preliminary evaluation using an isolated bovine liver.

Results: Gel strengths and gelation rates increased with the cross-link density between C and GP. These behaviors are more evident for C/GP solutions, which displayed a gel-like precipitation at 4°C. Furthermore, formulations with the lowest cross-link density between C and GP exhibited the best injectability due to a lower resistance to flow. The loading of the dye did not influence the gelation rate. ICG was not released from the hydrogels because of a strong electrostatic interaction between C and ICG. Ex vivo preliminary studies revealed that these injectable formulations remain in correspondence of the injected site.

Conclusions: The developed ICG-loaded hydrogels have the potential for intraoperative fluorescence imaging and local therapy of HCC as embolic agents. They form in situ compact gels and have a good potential for filling vessels and/or body cavities.     

Comparison of in situ gelling formulations for the oral delivery of cimetidine

Three liquid formulations with in situ gelling properties have been assessed for their potential for the oral delivery of cimetidine. The formulations were dilute solutions of: (a) enzyme-degraded xyloglucan, which form thermally reversible gels on warming to body temperature; (b) gellan gum and; (c) sodium alginate both containing complexed calcium ions that form gels when these ions are released in the acidic environment of the stomach. The in vitro release of cimetidine from gels of each of the compounds followed root-time kinetics over a period of 6 h. Plasma levels of cimetidine after oral administration to rabbits of each of the formulations were compared with those resulting from administration of a commercial cimetidine/alginate suspension with an identical drug loading. In vivo release characteristics of each of the in situ gelling formulations were similar to those of the commercial preparation.     

Injectable block copolymer hydrogels for sustained release of a PEGylated drug

The paper employs the spontaneous physical gelling property of a biodegradable polymer in water to prepare an injectable sustained release carrier for a PEGylated drug. A series of thermogelling PLGA-PEG-PLGA triblock copolymers were synthesized. The PEGylated camptothecin (CPT) was also prepared and employed as the model of a PEGylated drug, and the solubility of this hydrophobic drug was significantly enhanced to over 150mg/mL. The model drug was completely entrapped into the polymeric hydrogel, and the sustained release lasted for 1 month. The mechanism of the sustained release was diffusion-controlled at the first stage and then was the combination of diffusion and degradation at the late stage. In vivo anti-tumor tests in mice further confirmed the efficacy of the model PEGylated drug released from the hydrogel. This work also revealed the specificity of the PEGylated drug in such a kind of carrier systems by decreasing the critical gelling temperature and increasing the viscosity of the sol. Due to the very convenient drug formulation and highly tunable release rate, an injectable carrier platform for PEGylated drugs is thus set up.     

可注射硫酸延胡索总碱温度敏感型原位凝胶的 制备及其体外释药评析

 目的：研究可注射硫酸延胡索总碱原位凝胶的制备方法及其体外释药特性。方法：以泊洛沙姆407为载体材料制备注射用硫酸延胡索总碱温敏型原位凝胶制剂;采用高效液相色谱法测定释放介质中药物含量,考察凝胶体外释放特性;并对制剂进行评价。结果：原位凝胶最佳配方为：16%泊洛沙姆407、11%泊洛沙姆188、0.2%壳聚糖、0.5%硫酸延胡索总碱,该原位凝胶在室温下为流体,在体温下发生相转变形成凝胶;药物在48 h释放了88.4%,无突释现象,释药规律符合Higuchi方程;凝胶属Ostwald流体。结论：可注射硫酸延胡索总碱原位凝胶制备工艺简便,有良好的缓释作用,为研究中药植入型给药系统提供了依据。     

Controlled DNA Delivery Systems

Purpose. Genes are of increasing interest as pharmaceuticals, but current methods for long-term gene delivery are inadequate. Controlled release systems using biocompatible and/or biodegradable polymers offer many advantages over conventional gene delivery approaches. We have characterized systems for controlled delivery of DNA from implantable polymer matrices (EVAc: poly (ethylene-co-vinyl acetate)) and injectable microspheres (PLGA and PLA: poly (D, L-lactide-co-glycolide) copolymer and poly (L-lactide), respectively). Methods. Herring sperm DNA and bacteria phage DNA were encapsulated as a model system. Released DNA concentration was determined by fluoroassays. Agarose electrophoresis was used to determine the dependence of release rate on DNA size. The Green Fluorescent Protein (GFP) gene was used to determine the integrity and functionality of released DNA. Results. Both small and large DNA molecules (herring sperm DNA, 0.1–0.6 kb; GFP, 1.9 kb; DNA, 48.5 kb) were successfully encapsulated and released from EVAc matrices, and PLGA or PLA microspheres. The release from DNA-EVAc systems was diffusion-controlled. When co-encapsulated in the same matrix, the larger DNA was released more slowly than herring sperm; the rate of release scaled with the DNA diffusion coefficient in water. The chemical and biological integrity of released DNA was not changed. Conclusions. These low cost, and adjustable, controlled DNA delivery systems, using FDA-approved biocompatible/biodegradable and implantable/injectable materials, could be useful for in vivo gene delivery, such as DNA vaccination and gene therapy.     

Gelatin microspheres as a pulmonary delivery system: evaluation of salmon calcitonin absorption

The use of negatively and positively charged gelatin microspheres for pulmonary delivery of salmon calcitonin was examined in rats. The microspheres were prepared using acidic gelatin (isoelectric point (IEP):, 5.0) and basic gelatin (IEP, 9.0) for the negatively and positively charged microspheres, respectively. The average diameters of positively charged gelatin microspheres in the dry state were 3.4, 11.2, 22.5 and 71.5 microm, and that of negatively charged gelatin microspheres was 10.9 microm. Neither positively nor negatively charged gelatin microspheres underwent any degradation in pH 7.0 PBS and there was less than 8% degradation in bronchoalveolar lavage fluid (BALF) after 8 h. In in-vitro release studies in pH 7.0 PBS, salmon calcitonin was rapidly released from positively charged gelatin microspheres within 2 h, and its cumulative release was approximately 85%. In addition, the release profiles were not influenced by particle sizes. The release rates of salmon calcitonin from negatively charged gelatin microspheres were lower than that from positively charged gelatin microspheres. The cumulative release was approximately 40% after 2 h, but there was no evidence of any sustained release. The pulmonary absorption of salmon calcitonin from gelatin microspheres was estimated by measuring its hypocalcaemic effect in rats. The pharmacological availability after administration of salmon calcitonin in positively and negatively charged gelatin microspheres was significantly higher than that in pH 7.0 PBS. The pharmacological availability after administration of salmon calcitonin in positively charged gelatin microspheres was significantly higher than that in negatively charged gelatin microspheres. Administration of salmon calcitonin in positively charged gelatin microspheres with smaller particle sizes led to a higher pharmacological availability. The pharmacological availability after pulmonary administration of salmon calcitonin in positively charged gelatin microspheres with particle sizes of 3.4 and 11.2 microm was approximately 50%. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for pulmonary delivery of salmon calcitonin.     

Influence of end groups on in vitro release and biological activity of lysozyme from a phase-sensitive smart polymer-based in situ gel forming controlled release drug delivery system

Phase-sensitive in situ gel forming controlled release formulations of lysozyme were prepared using poly lactic acid (PLA) and/or poly glycolic acid (PGA) based polymers differing in end groups in addition to composition, and a solvent system consisting of various ratios of benzyl benzoate (BB) and benzyl alcohol (BA). The amount of lysozyme in the released samples was determined by measuring absorbance at 280 nm using suitable controls to nullify the effect of absorption of formulation degradation products. Biological activity of lysozyme was studied by an enzyme activity assay using Micrococcus lysodeikticus as substrate. Polymers bearing carboxylic acid end group were not soluble in 100% BB but polymers having ester end groups were soluble up to 27% (w/v) except polymer 4. A biphasic release profile consisting of slower first phase followed by faster second phase was observed. Formulations prepared from polymer with carboxylic acid groups showed significantly (p<0.05) lower burst release (4%) than those containing ester end groups (20-30%). However, formulations consisting of polymer with carboxylic acid end groups showed significantly (p<0.05) faster release rate of incorporated lysozyme, although the total amount released was less in comparison to the total amount released from formulations prepared using polymers containing ester end groups. The mean percentage specific enzyme activity (MPSEA) data were supported by the release profiles. In conclusion, polymer end groups may influence the release profiles of a protein from an in situ gel depot forming controlled release formulations.     

安乃近原位凝胶的制备及体外评价

目的 制备安乃近原位凝胶,延长安乃近在体内的作用时间.方法 以泊洛沙姆407(P407)和泊洛沙姆188(P188)作为载体材料,采用冷溶法制备安乃近原位凝胶,考察P407和P188的含量、亚硫酸氢钠及安乃近浓度对胶凝温度的影响,并用透析袋法考察凝胶的体外释药情况.结果 当P407浓度为23％、P188浓度为6％、亚硫酸氢钠的浓度为0.2％时,所制得的安乃近原位凝胶注射剂的胶凝温度合适,为35.3℃时,该凝胶在1h内的平均体外累积释放率为40.57％,较安乃近水溶液同时间内的累积释放率低50.39％.结论 安乃近原位凝胶的温敏性明显,其胶凝温度适合体内给药;安乃近原位凝胶对安乃近药物的释放显示出了明显的缓释效应,可以进一步用于临床.     

Development of albumin microspheres containing Sp H1-DNA complexes: A novel gene delivery system

This study investigated the stability and transfection efficiency of plasmid DNA (pDNA) and sea urchin sperm histone H1 (Sp H1) complexes embedded in albumin microsphere formulations. Sp H1 increased the stability and transfection efficiency of pDNA, while providing a favourable sustained pDNA release profile. Encapsulating Sp H1-complexed pDNA into albumin microspheres further protected the pDNA from physical stress and heparin treatment. When compared with free pDNA encapsulated in albumin microspheres, the Sp H1-pDNA microsphere formulations exhibited decreased hydrophilicity, slower pDNA release profiles, protection against heparin-induced degradation of embedded pDNA and increased stability against physical stress. These results indicate that complex formation of pDNA with Sp H1 facilitates intracellular DNA transfer and that albumin microspheres-Sp H1-pDNA gene delivery formulations are suitable for controlled-release delivery of pDNA while offering protection of the pDNA from degradation and maintaining pDNA biological activity.     

In situ gelling pectin formulations for oral drug delivery at high gastric pH

The aim of the study was to compare the gelation and drug release characteristics of formulations of pectin with high (31%) and low (9%) degrees of methoxylation over a wide pH range (pH 1.2-5.0). Dilute solutions of pectin (1.5%, w/v) containing complexed calcium ions formed gels in vitro at low pH (pH<2.5) as a consequence of cross-linking of the galacturonic chains by calcium ions released from the complex, but the efficiency of gelation was significantly reduced with increase of pH because of incomplete release of complexed Ca(++). Gelation of formulations of pectin with a degree of esterification of 9% (DE9) was observed over the pH range 2.5-5.0 in the presence of 1.6mM Ca(++), but was incomplete in formulations of pectin with a degree of esterification of 31% (DE31). A sustained release of ambroxol was observed following oral administration of pectin DE9 formulations to gastric-acidity controlled rabbits at pH 5.5-5.7 and visual observation of the stomach contents of these rabbits confirmed in situ gelation of these formulations. There was no evidence of in situ gelation of pectin DE31 formulations under these conditions and a rapid initial drug release was observed. Differences in gelling characteristics in this pH range were attributed to the greater susceptibility of low methoxylated pectin to cross-linking by di- and tri-valent ions present in the gastric juice. It is concluded that formulations of pectin with a low degree of esterification have potential application as in situ gelling vehicles for the sustained delivery of drugs following oral administration under conditions of high gastric pH.     

盐酸多西环素注射用缓释凝胶的体内释药研究

目的考察盐酸多西环素注射用缓释凝胶的体内释放特性及体内外相关性。方法将含药凝胶注射到家兔牙龈内 ,用HPLC法测定不同时间凝胶中的残余药量 ,从而计算药物的释放量。结果 7d盐酸多西环素的释放度达 99 7%。结论药物可缓释 7d ,体内外相关。     

In situ gel systems as 'smart' carriers for sustained ocular drug delivery

INTRODUCTION: In situ gel systems refer to a class of novel delivery vehicles, composed of natural, semisynthetic or synthetic polymers, which present the unique property of sol-gel conversion on receipt of biological stimulus. AREAS COVERED: The present review summarizes the latest developments in in situ gel technology, with regard to ophthalmic drug delivery. Starting with the mechanism of ocular absorption, the review expands on the fabrication of various polymeric in situ gel systems, made up of two or more polymers presenting multi-stimuli sensitivity, coupled with other interesting features, such as bio-adhesion, enhanced penetration or sustained release. Various key issues and challenges in this area have been addressed and critically analyzed. EXPERT OPINION: The advent of in situ gel systems has inaugurated a new transom for 'smart' ocular delivery. By virtue of possessing stimuli-responsive phase transition properties, these systems can easily be administered into the eye, similar to normal eye drops. Their unique gelling properties endow them with special features, such as prolonged retention at the site of administration, followed by sustained drug release. Despite the superiority of these systems as compared with conventional ophthalmic formulations, further investigations are necessary to address the toxicity issues, so as to minimize regulatory hurdles during commercialization.     

In situ forming hydrogels based on chitosan for drug delivery and tissue regeneration

In situ forming hydrogels with simple sol–gel transition are more practicable as injectable hydrogels for drug delivery and tissue regeneration. State-of-the-art in situ gelling systems can easily and efficiently be formed by different mechanisms in situ. Chitosan is a kind of natural polysaccharide that is widely exploited for biomedical applications due to its good biocompatibility, low immunogenicity and specific biological activities. Chitosan-based in situ gelling systems have already gained much attention as smart biomaterials in the development of several biomedical applications, such as for drug delivery systems and regeneration medicine. Herein, we review the typical in situ gelling systems based on chitosan and mechanisms involved in hydrogel forming, and report advances of chitosan-based in situ gels for the applications in drug delivery and tissue regeneration. Finally, development prospects of in situ forming hydrogels based on chitosan are also discussed in brief.     

In Vivo Properties of an In Situ Forming Gel for Parenteral Delivery of Macromolecular Drugs

Purpose. This study characterizes the in vivoproperties of an in situforming gel, comprising an IPC of water-soluble polymers, PMA and PEG, for sustained release of macromolecular drugs. Methods. 40, 50 or 60% w/v formulations were injected subcutaneously in a rat model either alone, or containing model macromolecules, 3A2-ATG-psODN or REV-psODN, to (i) determine the approximate gelling and residence time of the gel at the site of injection, (ii) assess the biological efficacy of the formulation using a MZ sleep time model, and (iii) demonstrate specificity of the sequence and selectivity of the psODNs by measuring changes in microsomal enzyme levels and urine volumes. Results. A sol to gel transition requires 15 min in vivo, and the 60% w/v IPC gel remains at the site of injection for up to 72 hr. The MZ sleep times and CYP3A2 expression due to 3A2-ATG-psODNs released from the gel are significantly different compared to that of REV-psODNs. Conclusions. The IPC solutions exhibit phase transformation in vivo, and demonstrate no evidence of toxicity. The pharmacological effects observed from the of release of 3A2-ATG-psODNs suggest that the formulation can entrap, protect, and sustain the delivery of macromolecules.     

Thermoreversible mucoadhesive ophthalmic in situ hydrogel: Design and optimization using a combination of polymers

The purpose of the study was to develop an optimized thermoreversible in situ gelling ophthalmic drug delivery system based on Pluronic F 127, containing moxifloxacin hydrochloride as a model drug. A 3(2) full factorial design was employed with two polymers: Pluronic F 68 and Gelrite as independent variables used in combination with Pluronic F 127. Gelation temperature, gel strength, bioadhesion force, viscosity and in vitro drug release after 1 and 10 h were selected as dependent variables. Pluronic F 68 loading with Pluronic F 127 was found to have a significant effect on gelation temperature of the formulation and to be of importance for gel formation at temperatures 33-36 °C. Gelrite loading showed a positive effect on bioadhesion force and gel strength and was also found helpful in controling the release rate of the drug. The quadratic mathematical model developed is applicable to predicting formulations with desired gelation temperature, gel strength, bioadhesion force and drug release properties.     

Gene Delivery in Tissue Engineering: A Photopolymer Platform to Coencapsulate Cells and Plasmid DNA

PURPOSE: Toward the ultimate goal of developing an engineered tissue capable of mimicking complex natural healing processes, we have designed a photopolymer platform that enables simultaneous encapsulation of cells and plasmid DNA in degradable hydrogels. Photopolymerization enables spatial and temporal control of gel formation under physiological conditions, but the presence of photoinitiator radicals poses challenges for DNA photoencapsulation. METHODS: The effects of photoinitiating conditions (ultraviolet light and photoinitiator radicals) on plasmid DNA were studied. Protection methods were identified. Plasmid DNA was photoencapsulated in photocrosslinked hydrogels, and the quantity and quality of the released DNA were assessed. Plasmid DNA was simultaneously entrapped (coencapsulated) with cells in hydrogels to assess in situ transfection. RESULTS: Experiments showed that in the absence of other species, plasmid DNA was sensitive to photoinitiator radicals, but the addition of transfection agents and/or antioxidants greatly reduced DNA damage by radicals. Encapsulated plasmid DNA was released from degradable, photocrosslinked hydrogels in active forms (supercoiled and relaxed plasmids) with an overall -60% recovery. Released DNA was capable of transfecting both plated and encapsulated cells. Encapsulated cells expressed the encoded gene of the coencapsulated plasmid as the polymer degraded. CONCLUSIONS: This photopolymerization platform allows for the creation of engineered tissues with enhanced control of cell behavior through the spatially and temporally controlled release of plasmid DNA.