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Evaluation of product viscosity, density and aeration on the dose delivery and accuracy for intravitreal injections with commonly used commercially available hypodermic 1 mL syringes. Six commercially available hypodermic 1 mL syringes with different specifications were used for the study. Syringes were filled with the test solutions with different densities and viscosities. Syringes were also subjected to shaking stress to introduce aeration in the test solutions in the presence of different surfactant concentrations with and without high antibody concentration. Target intravitreal volumes of 100 μL, 50 μL and 30 μL were tested to assess dosing accuracy in a controlled simulated administration setup using DIN ISO 11040-4 guidelines and Zwick/Roell Z010 TN instrument. With increasing product viscosity, higher volumes and hence doses were delivered especially for very low volumes like 50 μL and 30 μL. No impact of increasing product density was found on the delivered dose. The presence of surfactants or high protein concentration can lead to aeration, which also negatively affects the dose accuracy and precision. Formulation parameters like viscosity can have an impact on dose delivery using hypodermic syringes for intravitreal injections and on the resulting glide force.

Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g. 50 μL) and high injection forces. DPs in vials are typically transferred and injected using disposable polymer syringes, which can feature a silicone oil (SO) coating. In our syringe in-use study, we compared dead volume, total SO content and SO layer distributions of three IVT transfer injection syringes. We assessed multiple potential impact factors such as protein concentration, needle gauge, injection speed, surfactant type and the impact of the in-use hold time on sub-visible particle (SvP) formation and injection forces. Pronounced differences were observed between the syringes regarding SvP generation. Siliconized syringes showed higher SvP counts as compared to non-siliconized syringes. In some cases injection forces exceeded 20 N, which caused needles to burst off during injection. The syringes also showed relevant differences in total SO content and dead volume. In conclusion, specific consideration in the selection of an adequate transfer injection syringe are required. This includes extensive testing and characterization under intended and potential in-use conditions and the development of in-use handling procedures.

Gene therapy continues to hold promise in treating a variety of inherited and acquired diseases. The great majority of gene therapy trials rely on viral vectors for gene transduction because of their high efficiency. Non-viral vectors for gene delivery are receiving increasing attention for application in a wide variety of gene mediated therapies for humans. Polycationic polymers have been increasingly proposed as potential vectors because of their versatility. Rigidity, hydrophobicity/hydrophilicity, charge density, biodegradability, and molecular weight of the polymer chain are all parameters that in principle can be adjusted to achieve an optimal complexation with DNA. Polymers with repeating polyester bonds in the backbone are structurally versatile and biodegradable through hydrolysis, and possibly enzymatic digestion at the ester linkages under physiological conditions. These biodegradable polyesters are appealing for biological and pharmaceutical applications because of their potential bioc...

Biodegradable nano-polymeric carrier composed of polycaprolactone (PCL) and polyethylenimine (PEI) (BNPP) was successfully synthesized for the delivery of sh/siRNA in lung cancer cells. BNPP efficiently and safely delivered siRNA in lung cancer cells. BNPP-delivered Akt1 siRNA silenced Akt1 protein, and reduced the cancer cell survival, proliferation, malignancy and metastasis.

Gene therapy is a powerful approach in the treatment of a wide range of both inherited and acquired diseases. Nonviral delivery systems have been proposed as safer alternatives to viral vectors because they avoid the inherent immunogenicity and production problems that are seen when viral systems are used. Many cationic polymers, including high-molecular-weight polyethylenimine (PEI) have been widely studied as gene-delivery carriers, both, in vitro and in vivo. However, interest has recently developed in degradable polymeric systems. The advantage of degradable polymer is its low in-vivo cytotoxicity, which is a result of its easy elimination from the cells and body. Degradable polymer also enhances transfection of DNA or small interfering RNA (siRNA) for efficient gene expression or silencing, respectively. This review paper summarizes and discusses the recent advances with degradable PEIs, such as cross-linked and grafted PEIs for DNA and siRNA delivery.

Gene therapy is a powerful treatment for inborn and acquired diseases. The development of safe and effective gene delivery systems is a great challenge to make the human gene therapy a reality. Viral vectors have been commonly employed due to the high transfection efficiency, however, their application to the human body is often frustrated by immunogenicity, potential infectivity, complicated production, and inflammation. Non-viral vectors have been widely proposed as safer alternatives to viral vectors by reason of unique advantages such as less immune reaction against repeated administration, ease of synthesis, cell/tissue targeting, unrestricted plasmid size, and low cost. Among non-viral systems, cationic polymers have gained increasing attention because they can easily form self-assembly with DNA. Polyethylenimine (PEI) is one of the most popular cationic polymers investigated in non-viral gene therapy due to its ability to generate elevated levels of gene expression in vitro and in vivo comp...

Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(beta-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE-shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE-shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies.

Vectors are vital aspect of gene delivery system which decides the success of gene therapy. Efficient transfection with minimum or no toxicity, are two principal aims of any gene delivery system. In this our study, we rationally developed biodegradable water soluble poly(ßamino ester) (PAE) based on spermine (SPR) and poly (ethylene glycol) (PEG), by Michael-type addition reaction and further studied for its potential as a gene carrier. Confirmation of synthesized PAE was done by proton NMR spectroscopy. In gel retardation assay, the PAEs have shown good DNA binding ability over wide range of polyplexes. The addition of PEG over SPR resulted in a novel PAE with higher degree of safety and transfection efficiency as compared with polyethylenimine 25K (PEI) when studied in 293T human kidney carcinoma cells.

Novel, biodegradable poly(ester amine)s (PEAs) were synthesized using hydrophobic polycaprolactone diacrylate (PCLDA) and highly cationic polyethylenimine (PEI). This novel gene carrier can form stable DNA complexes with particle sizes around 200 nm, and showing excellent transfection efficiency and relatively low cytotoxicity compared with PEI 25K. Effect of hydrophobicity on transfection efficiency and cytotoxicity was profound and was relatively important parameter for the success of gene delivery.

Currently, the success of gene therapy is mainly limited due to the lack of effective vector systems. Although viral vectors are highly efficient in transfecting cells, undesirable complications limit their therapeutic applications. Chitosan has been investigated as a non-viral vector offering several advantages, such as biocompatibility, biodegradability and low toxicity with high cationic potential. However, the low transfection efficiency and low cell specificity of chitosan as a DNA carrier need to be overcome before undertaking clinical trials. The objective of this review is to summarize the use of chitosan and chitosan derivatives in gene therapy, and particularly the role of several factors for the enhancement of transfection efficiency and cell specificity in vitro, such as the degree of deacetylation and molecular weight of chitosan, pH, serum, charge ratio of chitosan to DNA and cell type on transfection efficiency, chemical modification. The administration of the chitosan derivative formulations in vivo is also included, and, the role of chitosan as a carrier of controlled release of DNA and small interfering RNA is described.

Cationic polymers that have shown significant promise as gene delivery agents are more effective than the state-of-the art, commercially available non-viral systems. Gene delivery in vivo involves interactions with the biophase (the effect site of drug) prior to reaching the target cells which complicates efforts to understand the mechanism of the delivery process. The ability to incorporate genetic materials such as DNA, RNA and siRNAs into functionalized nanoparticles demonstrates a new era. In this chapter, we highlight the basic overview of injectable polymeric gene carriers that have been reported as safe and successful vectors, their formulations and in vivo success thereof. In addition, we outline various strategies for designing polymeric carriers to overcome various biological barriers as successful gene delivery vectors.

Folate conjugated poly(ester amine) (PEA) was prepared by reaction of folic acid with PEAs based on polycaprolactone (PCL) and low molecular weight polyethylenimine (LMW-PEI) with PEG as a linker. This novel gene carrier showed excellent physicochemical properties and relatively low cytotoxicity compared with PEI 25K. It showed excellent transfection efficiency through folate receptor mediated endocytosis.

Biodegradable gene carrier, termed as PEI-alt-PEG, has been synthesized based on Michael addition reaction between lower Mw PEI and poly(ethylene glycol) (PEG) diacrylate and tested its potential of anti-metastatic cancer gene therapy by using anti-KITENIN short hairpin RNA. KITENIN is known to promote invasion of mouse colon adenocarcinoma in vivo. Intratumoral administration of anti-KITENIN shRNA-loaded PEI-alt-PEG nanoparticles has shown suppressed proliferlation and enhanced apoptosis signal in tumor compared to commercial available liposome, leading to delayed tumor growth.

To circumvent carrier related obstacles, we developed a biodegradable, folate conjugated poly (ester amine) (FP-PEA) that mediates high level folate receptor (FR) mediated endocytosis in vitro as well as in vivo. We report the efficacy of a therapeutic strategy that combines the potency of FP-PEA based on polycaprolactone (PCL) and low molecular weight polyethylenimine (LMW-PEI) with the tumor targeting potential of receptor mediated endocytosis. When tested on cells in culture, FP-PEA was found to retain high affinity for FR-positive cells compared with PEA without folate moiety (P-PEA). The FR specific activity of FP-PEA was drastically decreased in the presence of an excess free folic acid and very less significant transfection was detected against FR-negative cells. FP-PEA showed marked anti-tumor activity against FR-positive human KB tumors in nude mice with no evidence of toxicity during and after therapy using TAM67 gene. Furthermore, the therapeutic effect occurred in the apparent absence of weight loss or noticeable tumor apoptosis. In contrast, no significant anti-tumor activity was observed in P-PEA treated mice which were co dosed with an excess of FR, thus demonstrating the target specific gene delivery. Furthermore, anti-tumor activity with PEA without folic acid moiety (P-PEA) proved not to be effective against xenograft mice model with KB cells when administered at the same dose to that of FP-PEA. Taken together, these results indicate that FP-PEA is highly effective gene carrier capable of producing therapeutic benefit in xenograft mice model without any sign of toxicity.

Cationic polymers have been increasingly proposed as potential gene delivery vectors because of their versatility. In this paper, we focus on the characteristics of poly(ester amine)s (PEAs) as gene delivery carriers, degradation pattern as an essential parameter for reduced toxicity, classification based on its physicochemical properties followed by its success as efficient gene delivery carrier in vitro and in vivo. We also discuss the conjugation of ligands/charged groups to the side chain of the polyester in order to achieve receptor-mediated endocytosis as well as target-specific delivery of DNA. Capable of delivering exogenous genes to a cell nucleus, these cationic PEAs also serve as a valuable model to understand the important characteristics that render a polymer an effective gene carrier.