Physicochemical properties of pDNA/chitosan complexes as gene delivery systems

Successful gene therapy depends on the development of effective gene carriers. Naturally occurring chitosan has been employed widely as a non-viral gene carrier because of its low toxicity, low immunogenicity, biocompatibility, and biodegradability. In this review, we summarize the utilization of chitosan, modified chitosan, and chitosan-containing ternary complexes as gene carriers. In particular, we discuss the influence of the physicochemical features of pDNA/chitosan complexes on their functions, such as stability and gene transfer into cells.     

Carboxylated poly(glycerol methacrylate)s for doxorubicin delivery

Poly(glycerol methacrylate)s (PGOHMAs) were successfully synthesized via the hydrolysis of the epoxy groups on linear and/or star-shaped poly(glycidyl methacrylate)s (PGMAs). Further modification of the hydroxyl groups on PGOHMAs with succinic anhydride (SA) or 1,2-cyclohexanedicarboxylic anhydride (CDA) resulted in a new type of polyacid polymer, namely, PGOHMACOOH for short, which was then employed to prepare pH-sensitive assemblies using dialysis method. The carboxylated polymers were quite effective in the encapsulation of doxorubicin hydrochloride (DOX) by electrostatic interaction. Compared with poly(acrylic acid) (PAA), the star-shaped PGOHMA modified with CDA exhibited higher encapsulation efficiency and loading capacity, as well as better pH-responsive release profile. Scanning electron microscope images showed that the polymeric nanoparticles before and after encapsulation of DOX were spherical in shape. The encapsulation efficiency, loading capacity and release properties of these polymers were found to rely on their backbone architectures and the type of carboxylated functionalities. By fine-tuning these factors to achieve optimal properties, such type of polymeric materials holds promise as an attractive and effective drug delivery vehicle.     

Poly(methyl methacrylate) particulate carriers in drug delivery

Poly(methyl methacrylate) (PMMA) is one of the most widely explored biomedical materials because of its biocompatibility, and recent publications have shown an increasing interest in its applications as a drug carrier. PMMA-based particulate carriers (PMMA(P)) can be prepared either by polymerization methods or from pre-formed polymer-based techniques. Potential biomedical application of these particles includes their use as adjuvant for vaccines and carrier of many drugs as antibiotics and antioxidants via different routes of administration. Release of drugs from PMMA(P) occurs typically in a biphasic way with an incomplete drug release. To improve release profiles, recent strategies are focusing on increasing polymer hydrophilicity by synthesizing functionalized PMMA microspheres or by formulating PMMA composites with hydrophilic polymers. This review examines the current status of preparation techniques, drug release kinetics, biomedical applications and toxicity of these nano/micro PMMA-based particulate carriers.     

Formation and in-vitro evaluation of theophylline-loaded poly(methyl methacrylate) microspheres

Theophylline-loaded poly(methyl methacrylate) (PMM) microspheres were prepared by the solvent evaporation method. Increasing the drug to polymer ratio increased both the mean particle size of the microspheres and the release rate. Polyethylene glycol (PEG) 4000 was used to improve the release rate of theophylline from the microspheres. No marked effect was observed on particle size distribution of the microspheres as a function of PEG concentration but there was a pronounced effect on drug release. The different particle sizes of microspheres prepared from the same drug to polymer ratio showed no significant difference in drug content, indicating that the ratio between theophylline and PMM remained practically constant regardless of the size of microspheres. Release characteristics of the microspheres were influenced by drug to polymer ratio, the amount of PEG incorporated and the particle size of microspheres. The release rate was slightly higher in simulated gastric fluid than in simulated intestinal fluid. The release profiles of the drug were modified by mixing microspheres of different formulations in different ratios.     

Development of poly(hydroxyethyl methacrylate) nanogel for effective oral insulin delivery

Abstract

Because of uncomfortable, painful and even deleterious effects of daily injection of insulin, extensive efforts are being made worldwide for developing noninvasive drug delivery systems, especially via the oral route. In this study, we synthesized hydroxyethyl methacrylate (HEMA) nanogel via emulsion polymerization method. The morphology and stability of the nanogel were characterized by scanning electronic microscope and dynamic light scattering. In vivo results showed the soft HEMA nanogel had longer half-live in the body circulation and exhibited almost negligible uptake by the macrophage cells as compared with blank cells. For the FITC-dextran tracking for intestinal penetration, the results indicated that the FITC-dextran in the soft nanogel penetrated faster from the inner side of the abdominal segment, which explained why the soft HEMA nanogel could promote intestinal absorption of encapsulated insulin. In vivo delivery of insulin encapsulated in the soft HEMA nanogel sustained blood glucose control for 12?h, and the overall bioavailability of administrated insulin was much higher than free insulin. Our results showed that the insulin-loaded HEMA nanogel was able to efficiently control blood glucose as a delivery system, suggesting the HEMA nanogel using emulsion polymerization could be an alternative carrier for oral insulin delivery.     

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, were synthesized by Michael-type addition reaction between guanidine hydrochloride (CAR) or chlorhexidine (CHL) and N,N′-cystaminebisacrylamide (CBA). Previous studies have shown that both polymers had high transfection efficiencies as gene delivery carriers. In this study, we investigated the nucleolus localization abilities and cellular internalization pathways of these two polymers in gene delivery. Each polymer condensed plasmid DNA (pDNA) and formed nanoparticle complexes, and then their transfection studies were performed in MCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection, and their transfection efficiencies were significantly improved when transfection was performed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNA was inhibited by chlorpromazine, and cell endosomes were disrupted after being exposed to CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin- and caveolae-independent mechanism.     

Dexamethasone transport and ocular delivery from poly(hydroxyethyl methacrylate) gels

We explore ocular delivery of dexamethasone (DX) via poly(hydroxyethyl methacrylate) (PHEMA) contact lenses, which are known to have a much higher bioavailability in comparison to eye drops. Three derivatives of dexamethasone (dexamethasone 21-disodium phosphate (DXP), dexamethasone, and dexamethasone 21-acetate (DXA)) are explored. These drugs are loaded in the gels by soaking in aqueous or ethanol solutions, and also by direct addition of the drug to the polymerizing mixture. Dynamic drug concentrations in the aqueous phase are monitored both in loading and release experiments. The data is utilized to determine the partition coefficients and the mean diffusivity, which includes contributions from both bulk and surface diffusion. Finally we utilize the transport model to predict the bioavailability of the three forms of dexamethasone for drug delivery via contact lenses. The transport of each of the drug is diffusion limited with diffusivities of 1.08 x 10(-11) and 1.16 x 10(-11) m(2)/s for DX and DXA, respectively. The diffusivities of DXP depend on concentration and on ionic strength, and are much smaller than those for DX and DXP. The bioavailability for delivery of these drugs via contact lenses is much higher than that for drops, and the bioavailability is the highest for DXA.     

Chitosan-functionalised poly(2-hydroxyethyl methacrylate) core-shell microgels as drug delivery carriers: salicylic acid loading and release

This work presents the evaluation of chitosan-functionalised poly(2-hydroxyethyl methacrylate) (CS/PHEMA) core-shell microgels as drug delivery carriers. CS/PHEMA microgels were prepared by emulsifier-free emulsion polymerisation with N,N?′-methylenebisacrylamide (MBA) as a crosslinker. The study on drug loading, using salicylic acid (SA) as a model drug, was performed. The results showed that the encapsulation efficiency (EE) increased as drug-to-microgel ratio was increased. Higher EE can be achieved with the increase in degree of crosslinking, by increasing the amount of MBA from 0.01?g to 0.03?g. In addition, the highest EE (61.1%) was observed at pH 3. The highest release of SA (60%) was noticed at pH 2.4, while the lowest one (49.4%) was obtained at pH 7.4. Moreover, the highest release of SA was enhanced by the presence of 0.2 M NaCl. The pH- and ionic-sensitivity of CS/PHEMA could be useful as a sustained release delivery device, especially for oral delivery.     

One-step preparation of amino-PEG modified poly(methyl methacrylate) microchips for electrophoretic separation of biomolecules

A simple method for a chemical surface modification of poly(methyl methacrylate) (PMMA) microchips with amino-poly(ethyleneglycol) (PEG–NH₂) by nucleophilic addition–elimination reaction was developed to improve the separation efficiency and analytical reproducibility in a microchip electrophoresis (MCE) analysis of biomolecules such as proteins and enantiomers. In our procedure, the PEG chains were robustly immobilized only by introducing an aqueous solution of PEG–NH₂ into the PMMA microchannel. The electroosmotic mobilities on the modified chips remained almost constant during 35 days with 37 runs without any recoating. The PEG–NH₂ modified chip provided a fast, reproducible, efficient MCE separation of proteins with a wide variety of isoelectric points within 15 s. Furthermore, the application of the modified chip to affinity electrophoresis using bovine serum albumin gave a good chiral separation of amino acids.     

In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides

The efficacy of antisense oligodeoxynucleotides (AsODNs) is compromised by their poor stability in biological fluids and the inefficient cellular uptake due to their size and negative charge. Since chemical modifications of these molecules have resulted in a number of non-antisense activities, incorporation into particulate delivery systems has offered a promising alternative. The aim of this study was to evaluate various poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for AsODN entrapment and delivery. PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. The influence of formulation parameters such as PLGA concentration and volume ratio of internal aqueous phase volume (Va1) to organic phase volume (Vo) to external aqueous phase volume (Va2) on particle size, polydispersity index (PDI) and zeta potential (ZP) was investigated using a full factorial study. The particle size increased with increasing PLGA concentrations and volume ratios, with an interaction detectable between the two factors. AsODN entrapment efficiencies ranged between 49.97% and 54.95% with no significant difference between various formulations. By fitting the in vitro release profiles to a dual first order release model it was shown that the AsODN release occurred via two processes: a diffusion controlled process in the early phase (25 to 32% within one day) and a PLGA degradation process in the latter (39 to 70% after 14 days). Cellular uptake studies using primary corneal epithelial cells suggested active transport of nanoparticles via endocytosis. PLGA nanoparticles therefore show potential to successfully entrap AsODNs, transport them into cells and release them over time due to polymer erosion.     

In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides

The efficacy of antisense oligodeoxynucleotides (AsODNs) is compromised by their poor stability in biological fluids and the inefficient cellular uptake due to their size and negative charge. Since chemical modifications of these molecules have resulted in a number of non-antisense activities, incorporation into particulate delivery systems has offered a promising alternative. The aim of this study was to evaluate various poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for AsODN entrapment and delivery. PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. The influence of formulation parameters such as PLGA concentration and volume ratio of internal aqueous phase volume (Va1) to organic phase volume (Vo) to external aqueous phase volume (Va2) on particle size, polydispersity index (PDI) and zeta potential (ZP) was investigated using a full factorial study. The particle size increased with increasing PLGA concentrations and volume ratios, with an interaction detectable between the two factors. AsODN entrapment efficiencies ranged between 49.97% and 54.95% with no significant difference between various formulations. By fitting the in vitro release profiles to a dual first order release model it was shown that the AsODN release occurred via two processes: a diffusion controlled process in the early phase (25 to 32% within one day) and a PLGA degradation process in the latter (39 to 70% after 14 days). Cellular uptake studies using primary corneal epithelial cells suggested active transport of nanoparticles via endocytosis. PLGA nanoparticles therefore show potential to successfully entrap AsODNs, transport them into cells and release them over time due to polymer erosion.     

Investigation of triacetin effect on indomethacin release from poly(methyl methacrylate) microspheres: evaluation of interactions using FT-IR and NMR spectroscopies

The influx of medicines from different sources into healthcare systems of developing countries presents a challenge to monitor their origin and quality. The absence of a repository of reference samples or spectra prevents the analysis of tablets by direct comparison. A set of paracetamol tablets purchased in Malaysian pharmacies were compared to a similar set of sample purchased in the UK using near-infrared spectroscopy (NIRS). Additional samples of products containing ibuprofen or paracetamol in combination with other actives were added to the study as negative controls. NIR spectra of the samples were acquired and compared by using multivariate modeling and classification algorithms (PCA/SIMCA) and stored in a spectral database. All analysed paracetamol samples contained the purported active ingredient with only 1 out of 20 batches excluded from the 95% confidence interval, while the negative controls were clearly classified as outliers of the set. Although the substandard products were not detected in the purchased sample set, our results indicated variability in the quality of the Malaysian tablets. A database of spectra was created and search methods were evaluated for correct identification of tablets. The approach presented here can be further developed as a method for identifying substandard pharmaceutical products.     

Increased in vitro leishmanicidal activity of octyl gallate loaded poly(methyl methacrylate) nanoparticles

The current paucity of effective and affordable drugs for the treatment of leishmaniasis renders the search for new therapeutic alternatives a priority. Gallic acid-related compounds display anti-parasitic activities and their incorporation into drug carrier systems, such as polymeric nanoparticles may be a viable alternative for leishmaniasis treatment. Therefore, this study focused on the synthesis and characterization of octyl gallate (G8) loaded poly(methyl methacrylate) (PMMA) nanoparticles via miniemulsion polymerization in order to increase the leishmanicidal activity of this compound. G8 loaded PMMA nanoparticles presented a spherical morphology with a mean size of 108?nm, a negatively charged surface (?33?±?5?mV) and high encapsulation efficiency (83% ± 5). Fourier-transform infrared spectroscopy and X-ray diffraction analysis confirmed that G8 was encapsulated in PMMA nanoparticles and presented a biphasic release profile. The G8 loaded PMMA nanoparticles did not present cytotoxic effect on human red blood cells. G8 loaded PMMA nanoparticles displayed a leishmanicidal activity almost three times higher than free G8 while the cytotoxic activity against human THP-1 cells remained unchanged.     

Reduction of the uptake by a macrophagic cell line of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)

Amphiphilic and fluorescent covalently labelled core-shell nanoparticles based on poly(methyl methacrylate) (PMMA), were prepared by random copolymerisation of N-Vinyl carbazole (NVC) with MMA, initiated on polysaccharidic radicals, yielding diblock copolymers of either dextran-P(MMA-NVC) (Nanodex* particles), or heparin-P(MMA-NVC) (Nanohep* particles). Nanoparticles made from random copolymers of P(MMA-NVC) (PMMA*) were used as controls. The interactions between particles and a J774A1 murine macrophage-like cell line were quantified by direct measurement of the cell-associated fluorescence. The association with the cells occurred within 30 min. Nanodex* and Nanohep* showed considerably less association than the control PMMA* particles. Some of the particle uptake could be attributed to phagocytosis, but more than 50% of the cell-associated fluorescence persisted at low temperature or in the presence of cytochalasin B. The results suggest that both the adsorption and the internalisation processes can be inhibited by the presence of the polysaccharide chains. In conclusion, these results confirm that nanoparticles prepared with heparin or dextran chains on their surface, probably in a brush-like configuration, show "stealth" properties in vitro as had previously been observed in vivo. If this biomimetic approach can also be applied to biodegradable polymers, these systems would provide at least an alternative to PEG-modified particles as long-circulating drug carriers systems or imaging agents.     

PEGylated quaternized copolymer/DNA complexes for gene delivery

The aim of this study was to improve the colloidal stability, decrease unspecific interactions with cells and blood components of a novel gene delivery system composed of epsilon-caprolactone and quaternized epsilon-caprolactone. For this purpose, diblock 50/50 copolymer was used to generate complexes with DNA by either the solvent evaporation technique and by dialysis. The size, surface charge and degree of interaction of the plasmid-loaded formulations were measured. Then, polyplexes were combined with a poly(CL)-b-PEG copolymer to create a hydrophilic corona on the surface of the complexes. The cytotoxicity, transfection efficiency and cellular uptake of polyplexes and their association with PEG were evaluated on HeLa cells. The dialysis method did not allow to reduce the size of complexes as compared to the solvent evaporation method. The zeta potential of polyplexes became positive from a charge ratio of 4. The degree of interaction of copolymer with plasmid DNA was very high. Cytotoxicity and transfection efficiency were found to be comparable to polyethylenimine 50 kDa. Association of polyplexes with poly(CL)-b-PEG copolymer led to a small increase in particle size and a sharp decrease of charge surface. Cytotoxicity, transfection efficiency and cellular uptake were significantly reduced relative to unshielded copolymer/DNA complexes. The PEGylated formulations may be an attractive approach for an in vivo application.     

Microspheres of essential oil in polylactic acid and poly(methyl methacrylate) matrices and their blends

This study is focussed on micro-encapsulation of essential oils in polylactic acid (PLA) and a poly(methyl methacrylate) (PMMA) matrix as well as blends of the same. Microspheres were prepared by the solvent evaporation technique and characterised by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier transform infra-red spectroscopy (FTIR). The encapsulation efficiencies and release profiles of the essential oils were studied by gas chromatography mass spectrometry (GC-MS) and head-space solid-phase microextraction GC-MS, respectively. Furthermore, the microspheres were tested for antibacterial activity against both Gram-negative and Gram-positive bacterial strains.

The results showed that the microspheres compositions (PLA/PMMA ratio) have significant effect on their characteristics. The process adopted for preparing the microspheres promoted formation of spherical particles at the sizes of 1.5–9.5?µm. The highest encapsulation efficiency of the prepared microspheres was observed in systems consisting of linalool (81.10?±?10.0?wt. % for PLA system and 76.0?±?3.3?wt. % for PMMA system). Confirmation was also made that the release rate of the microspheres was affected by the size of the same.     

Poly(amidoamine) dendrimer complexes as a platform for gene delivery

Introduction: Gene therapy is one of the most effective ways to treat major infectious diseases, cancer and genetic disorders. It is based on several viral and non-viral systems for nucleic acid delivery. The number of clinical trials based on application of non-viral drug and gene delivery systems is rapidly increasing.

Areas covered: This review discusses and summarizes recent advances in poly(amidoamine) dendrimers as effective gene carriers in vitro and in vivo, and their advantages and disadvantages relative to viral vectors and other non-viral systems (liposomes, linear polymers) are considered.

Expert opinion: In this regard, dendrimers are non-immunogenic and have the highest efficiency of transfection among other non-viral systems, and none of the drawbacks characteristic for viral systems. The toxicity of dendrimers both in vitro and in vivo is an important question that has been addressed on many occasions. Several non-toxic and efficient multifunctional dendrimer-based conjugates for gene delivery, along with modifications to improve transfection efficiency while decreasing cytotoxicity, are discussed. Twelve paradigms that affected the development of dendrimer-based gene delivery are described. The conclusion is that dendrimers are promising candidates for gene delivery, but this is just the beginning and further studies are required before using them in human gene therapy.