Cryoprotection in Human Mesenchymal Stromal/Stem Cells: Synergistic Impact of Urea and Glucose.

Standard freezing protocols of clinically relevant cell lines commonly employ agents such as fetal bovine serum and dimethyl sulfoxide, which are a potential concern from both a regulatory and a patient safety perspective. The aim of this work was to develop formulations with safe and well tolerated excipients for the (cryo-) preservation of cell therapy products. We evaluated the cryoprotective capabilities of urea and glucose through measurements of cell metabolic activity. Freezing of clinically relevant human mesenchymal stromal/stem cells and human dermal fibroblasts at ≤ - 65°C at equimolar ratios of urea and glucose resulted in comparable viabilities to established dimethyl sulfoxide. Pre-incubation of human mesenchymal stromal/stem cells in trehalose and addition of mannitol and sucrose to the formulation further enhanced cell viability after freeze-thaw stress. Other cell types assessed (A549 and SK-N-AS) could not satisfactorily be preserved with urea and glucose, highlighting the need for tailored formulations to sustain acceptable cryopreservation.

Evaluation of In Vitro Tools to Predict the In Vivo Absorption of Biopharmaceuticals Following Subcutaneous Administration.

PURPOSE: For injectable biopharmaceuticals, the subcutaneous route of administration is increasingly preferred over intravenous administration. However, one of the challenges in the development of subcutaneously administered biopharmaceuticals is a reduced bioavailability, which is difficult to predict. Since animal models do not reliably reflect bioavailability in patients, in vitro models could help to develop drug candidates. The purpose of this study was to evaluate a versatile set of in vitro tools for their suitability to predict bioavailability of biopharmaceuticals after subcutaneous administration. METHODS: We examined seven commercially available biopharmaceuticals using three instruments, i.e., the Subcutaneous Injection Site Simulator (Scissor), the Osmomat 050, and a dialysis system using three artificial extracellular matrices, two dissolution apparatuses, i.e., the USP4 and the USP7, and two evaluation tools, i.e., the affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) and the Developability Index (DI). Results were evaluated for their usefulness to predict the bioavailability and other pharmacokinetic parameters in humans using the Pearson correlation. RESULTS: None of the tested instruments and methods could reliably approximate bioavailability. Only pressure values derived with the Osmomat 050 instrument correlated with Cmax with a Pearson correlation coefficient greater than 0.8. CONCLUSION: No single in vitro method confidently predicted the bioavailability in humans. We only found a correlation to maximum plasma concentration values for one of the tested approaches. However, a more focused evaluation would be necessary to confirm our findings and test combinations of orthogonal methods that may improve the confidence of such a prediction.

Challenges for Cell-Based Medicinal Products From a Pharmaceutical Product Perspective.

Advanced therapy medicinal products (ATMPs), such as somatic cell-therapy medicinal products or tissue-engineered products for human use, offer new and potentially curative opportunities to treat yet untreatable diseases or disorders. For cell-therapy medicinal products (CBMPs), multiple stability and quality challenges exist and relate to the cellular composition and unstable nature of these parenteral preparations. It is the aim of this review to discuss open questions and problems associated with the development, manufacturing and testing of CBMPs from a pharmaceutical drug product perspective. This includes safety, storage and handling, particulates, the choice of container closure systems and integrity. Analytical methods commonly used to evaluate the quality of the final CBMP to ensure patient's safety will be discussed. Particulate contamination in final products deserve special attention since CBMPs cannot be sterile filtered. Visible and sub-visible particles may represent environmental contaminations or may form during storage. They may be introduced from processing materials such as single use product contact materials, ancillary materials, or any components such as primary packaging used for the final product. Currently available analytical methods for detecting particulates may not be easily applicable to CBMPs due to their inherent particulate nature and appearance.

Role of Formulation Parameters on Intravitreal Dosing Accuracy Using 1 mL Hypodermic Syringes.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Formulation parameters like viscosity can have an impact on dose delivery using hypodermic syringes for intravitreal injections and on the resulting glide force.

Characterization of Polymeric Syringes Used for Intravitreal Injection.

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.

Subvisible Particulate Contamination in Cell Therapy Products-Can We Distinguish?

Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products.

Use of the permitted daily exposure (PDE) concept for contaminants of intravitreal (IVT) drugs in multipurpose manufacturing facilities.

A toxicological evaluation to determine the product specific permitted daily exposure (PDE) value is an accepted method to determine a safe limit for the carry-over of product residues in multipurpose manufacturing facilities. The PDE calculation for intravitreal (IVT) injection of small and large molecular weight (MW) drugs follows the guiding principles set for systemic administration. However, there are specific differences with respect to the volume administered with IVT administration, pharmacokinetic and pharmacodynamics (PK-PD) parameters and potential for toxicity. In this publication, we have proposed a method to derive PDEIVT in the presence of IVT dose. In the absence of an IVT dose we have a proposed default extrapolationof the systemic PDE for intravenous (IV) administration to the PDEIVT dose by applying a factor of 500 based on comparison of the volume of vitreous humour with the plasma volume, as well as provided examples for PK-PD and toxicity considerations.

Advancements in Understanding Immunogenicity of Biotherapeutics in the Intraocular Space.

Therapeutic breakthroughs in a number of retinal degenerative diseases have come about through the development of biotherapeutics administered directly into the eye. As a consequence of their use, we have gained more insight into the immune privileged status of the eye and the various considerations that development, manufacturing, and use of these drugs require. It has been observed that therapeutic proteins injected into the vitreous can elicit an immune response resulting in the production of anti-drug antibodies (ADAs) which can have clinical consequences. This review includes discussion of the anatomy, physiology, and specific area of the eye that are targeted for drug administration. The various immunologic mechanisms involved in the immune responses to intraocularly administered protein are discussed. This review entails discussion on chemistry, manufacturing, and control (CMC) and formulation-related issues that may influence the risk of immunogenicity. Based on the available immunogenicity profile of the marketed intraocular drugs and their reported adverse events, the animal models and the translational gap from animals to human are discussed. Thus, the objective of this review article is to assess the factors that influence immunogenicity in relation to intraocular administration and the steps taken for mitigating immunogenicity risks.

Prediction of intraocular antibody drug stability using ex-vivo ocular model.

Following intravitreal (IVT) injection, therapeutic proteins get exposed to physiological pH, temperature and components in the vitreous humor (VH) for a significantly long time. Therefore, it is of interest to study the stability of the proteins in the VH. However, the challenge posed by the isolated VH (such as pH shift upon isolation and incubation due to the formation of smaller molecular weight (MW) degradation products) can result in artefacts when investigating protein stability in relevance for the actual in vivo situation. In this current study, an ex-vivo intravitreal horizontal stability model (ExVit-HS) has been successfully developed and an assessment of long-term stability of a bi-specific monoclonal antibody (mAb) drug in the isolated VH for 3months at physiological conditions has been conducted. The stability assessment was performed using various analytical techniques such as microscopy, UV visible for protein content, target binding ELISA, Differential Scanning Calorimetry (DSC), Capillary-electrophoresis-SDS, Size Exclusion (SEC) and Ion-exchange chromatography (IEC) and SPR-Biacore. The results show that the ExVit-HS model was successful in maintaining the VH at physiological conditions and retained a majority of protein in the VH-compartment throughout the study period. The mAb exhibited significantly less fragmentation in the VH relative to the PBS control; however, chemical stability of the mAb was equally compromised in VH and PBS. Interestingly, in the PBS control, mAb showed a rapid linear loss in the binding affinity. The loss in binding was almost 20% higher compared to that in VH after 3months. The results clearly suggest that the mAb has different degradation kinetics in the VH compared to PBS. These results suggest that it is beneficial to investigate the stability in the VH for drugs intended for IVT injection and that are expected longer residence times in the VH. The studies show that the ExVit-HS model may become a valuable tool for evaluating stability of protein drugs and other molecules following IVT injection.

Evaluation of protein drug stability with vitreous humor in a novel ex-vivo intraocular model.

The stability of protein therapeutics during the residence time in the vitreous humor (VH) is an important consideration for intra ocular treatment and can possibly impact therapeutic efficacy and/or treatment intervals. Unavailability of the reliable Ex-vivo intravitreal (ExVit) model to estimate protein stability following IVT has driven the research focus to develop such model which can facilitate protein stability estimation before in-vivo experiments. In this manuscript, we have developed and evaluated three ExVit models, namely, ExVit static, semi-dynamic and dynamic. These models were utilized and compared when studying the in-vitro stability of model protein formulations under simulated intraocular conditions using porcine vitreous humor (VH). The ExVit static model exhibited significant precipitation and aggregation of proteins, most likely due to pH change occurred in the VH after isolation. The semi-dynamic model assessed was composed of two compartments i.e., VH- and buffer-compartment which has effectively stabilized the pH of the VH and facilitated the migration of VH degradation products. However, some limitations related to investigation of long-term protein stability were also observed with semi-dynamic model. The dynamic model developed, was comprised of three diffusion controlling barriers (two diffusion controlling membranes and a gel-matrix), which allowed modulation of the diffusion rate of macromolecules. The ability of dynamic model to modulate protein retention time in the VH will overcome the challenges faced by the semi-dynamic model such as long-term stability evaluation.

Intratumoral administration of anti-KITENIN shRNA-loaded PEI-alt-PEG nanoparticles suppressed colon carcinoma established subcutaneously in mice.

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.

Folate conjugated poly(ester amine) for lung cancer therapy.

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 nano-polymeric system for efficient Akt1 siRNA delivery.

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.

Biodegradable polymer-mediated sh/siRNA delivery for cancer studies.

Discovery of RNA interference (RNAi)-mediated specific gene silencing has raised hope for cancer therapy. Unfortunately, the execution of RNAi by delivering small-interfering RNA (siRNA) or small hairpin RNA (shRNA) remains a prime challenge. A methodical evaluation of cationic polymers in RNAi-based cancer studies may offer a promising solution to this problem. In this chapter, we report the methodologies for comprehensive characterization of a biodegradable polymeric system for sh/siRNA delivery in cancer studies. The chapter will describe synthesis, characterization, and optimization of biodegradable poly (beta-amino ester) for sh/siRNA delivery. The protocols are provided for shRNA and siRNA complex preparation, stability and morphology study. Also, detailed methods are provided for the intracellular tracking and transfection of sh/siRNA using polymeric carrier. In addition, step-wise information is provided for the in vitro silencing of oncoprotein to study important cancer properties, including proliferation, malignancy, and metastasis of cancer cells.

Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses.

BACKGROUND: Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA. RESULTS: In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220+ cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice. CONCLUSION: These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

The therapeutic efficiency of FP-PEA/TAM67 gene complexes via folate receptor-mediated endocytosis in a xenograft mice model.

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.

The suppression of lung tumorigenesis by aerosol-delivered folate-chitosan-graft-polyethylenimine/Akt1 shRNA complexes through the Akt signaling pathway.

RNA interference (RNAi) represents a promising new approach to the inhibition of gene expression in vitro and in vivo, and has therapeutic potential for human diseases. Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNAi studies. Here we report the development of a new polymeric gene carrier for cancer cell-targeting, designed to enhance the intracellular delivery of shRNA and reduce cytotoxicity. Folate-chitosan-graft-polyethylenimine (FC-g-PEI) copolymer was prepared by an imine reaction between periodate-oxidized folate-chitosan (FC) and low molecular weight polyethylenimine (PEI). FC-g-PEI copolymer was investigated as a potential cancer cell-targeting gene carrier. The composition of FC-g-PEI was characterized using (1)H nuclear magnetic resonance ((1)H NMR), and particle size and zeta potential of FC-g-PEI/shRNA complexes were measured using dynamic light scattering (DLS). FC-g-PEI showed good shRNA condensation ability and high protection of shRNA from nuclease attack. It also exhibited lower cytotoxicity compared to PEI 25K control, and showed good cancer cell-targeting ability. Furthermore, aerosol delivery of FC-g-PEI/Akt1 shRNA complexes suppressed lung tumorigenesis in a urethane-induced lung cancer model mouse through the Akt signaling pathway. Together, these results suggest that FC-g-PEI may be useful for shRNA-based gene therapy.

Biodegradable poly(ester amine)s for gene delivery applications.

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.

Mannosylated chitosan-graft-polyethylenimine as a gene carrier for Raw 264.7 cell targeting.

Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic genes. Among non-viral vectors, chitosans have been proposed as alternative, biocompatible cationic polymers for non-viral gene delivery. However, the low transfection efficiency and low specificity of chitosan needs to be addressed prior to clinical application. In this study, mannosylated chitosan-graft-polyethylenimine (Man-CHI-g-PEI) copolymer was prepared by thiourea reaction between the isothiocyanate group of mannopyranosylphenylisothiocyanate and the amine groups of chitosan-graft-PEI (CHI-g-PEI) for targeting into antigen presenting cells (APCs) having mannose receptors. The composition and molecular weight were characterized using (1)H NMR and GPC, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. Man-CHI-g-PEI showed good DNA binding ability and high protection of DNA from nuclease attack and had low cytotoxicity compared with PEI 25K. The transfection efficiency of Man-CHI-g-PEI/DNA complexes into the Raw 264.7 macrophage cell line, which has mannose receptors, was higher than CHI-g-PEI itself as well as PEI 25K, indicating Man-CHI-g-PEI can be used as an APCs' targeting gene delivery carrier.

Chitosan-graft-polyethylenimine for Akt1 siRNA delivery to lung cancer cells.

Efficient delivery of small interfering RNA (siRNA) remains a challenging task in RNA interference (RNAi) studies. In this study, we used chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer composed of chitosan and low molecular weight polyethylenimine (PEI) for the delivery of siRNA. The CHI-g-PEI carrier formed stable complexes with siRNA with compact spherical morphology. CHI-g-PEI delivered EGFP siRNA (siGFP) silenced EGFP expression nearly 2.5 folds higher than PEI25K at 50 pM siGFP concentration. Cell viability was found to be 2 folds high with CHI-g-PEI carrier than PEI25K. Also, our CHI-g-PEI carrier efficiently delivered Akt1 siRNA (siAkt) and thereby silenced onco-protein Akt1. Silencing of this crucial cell survival protein significantly reduced the lung cancer cell survival and proliferation. Additionally, Akt1 protein knock-down decreased A549 cell malignancy and metastasis. These findings suggest that the CHI-g-PEI carrier efficiently and safely delivered siRNA. Moreover, CHI-g-PEI mediated Akt1 siRNA delivery may emerge as a viable approach for lung cancer treatment.