Aerosolization of poly(sulfobetaine) microparticles that encapsulate therapeutic antibodies.

Pulmonary delivery of protein therapeutics poses significant challenges that have not been well addressed in the research literature or practice. In fact, there is currently only one commercial protein therapeutic that is delivered through aerosolization and inhalation. In this study, we propose a drug delivery strategy that enables a high-concentration dosage for the pulmonary delivery of antibodies as an aerosolizable solid powder with desired stability. We utilized zwitterionic polymers for their promising properties as drug delivery vehicles and synthesized swellable, biodegradable poly(sulfo-betaine) (pSB) microparticles. The microparticles were loaded with Immunoglobulin G (IgG) as a model antibody. We quantified the microparticle size and morphology, and the particles were found to have an average diameter of 1.6 µm, falling within the optimal range (~1-5 µm) for pulmonary drug delivery. In addition, we quantified the impact of the crosslinker to monomer ratio on particle morphology and drug loading capacity. The results showed that there is a trade-off between desired morphology and drug loading capacity as the crosslinker density increases. In addition, the particles were aerosolized, and our data indicated that the particles remained intact and retained their initial morphology and size after aerosolization. The combination of morphology, particle size, antibody loading capacity, low cytotoxicity, and ease of aerosolization support the potential use of these particles for pulmonary delivery of protein therapeutics.

Biodegradable zwitterionic poly(carboxybetaine) microgel for sustained delivery of antibodies with extended stability and preserved function.

Many recent innovative treatments are based on monoclonal antibodies (mAbs) and other protein therapies. Nevertheless, sustained subcutaneous, oral or pulmonary delivery of such therapeutics is limited by the poor stability, short half-life, and non-specific interactions between the antibody (Ab) and delivery vehicle. Protein stabilizers (osmolytes) such as carboxybetaine can prevent non-specific interactions within proteins. In this work, a biodegradable zwitterionic poly(carboxybetaine), pCB, based microgel covalently crosslinked with tetra(ethylene glycol) diacrylate (TTEGDA) was synthesized for Ab encapsulation. The resulting microgels were characterized via FTIR, diffusion NMR, small-angle neutron scattering (SANS), and cell culture studies. The microgels were found to contain up to 97.5% water content and showed excellent degradability that can be tuned with crosslinking density. Cell compatibility of the microgel was studied by assessing the toxicity and immunogenicity in vitro. Cells exposed to microgel showed complete viability and no pro-inflammatory secretion of interleukin 6 (IL6) or tumor necrosis factor-alpha (TNFα). Microgel was loaded with Immunoglobulin G (as a model Ab), using a post-fabrication loading technique, and Ab sustained release from microgels of varying crosslinking densities was studied. The released Abs (especially from the high crosslinked microgels) proved to be completely active and able to bind with Ab receptors. This study opens a new horizon for scientists to use such a platform for local delivery of Abs to the desired target with minimized non-specific interactions.

Surface Modification of Adenovirus Vector to Improve Immunogenicity and Tropism.

Although adenovirus is a popular vector for delivering genes, there are several drawbacks that limit its effectiveness, including tropism and both the innate and adaptive immune responses. One approach that has been used to ameliorate these drawbacks is PEGylation of the virus with subsequent modification to add functional moieties for the purpose of cell targeting or enhancing infection. Here, we describe a general approach for PEGylating adenovirus and conjugating cell-penetrating peptides to the surface of the virus to impart the ability to transduce CAR-negative cells.

Fabricating an electroactive injectable hydrogel based on pluronic-chitosan/aniline-pentamer containing angiogenic factor for functional repair of the hippocampus ischemia rat model.

The hippocampus, a critical cerebral region involved in learning and memory formation, is especially vulnerable to ischemic defect. Here, we developed an injectable electroactive hydrogel based on pluronic-chitosan/aniline-pentamer with proper conductivity around 10-4 S/cm to achieve the functional repair of the hippocampus following the ischemic defect. FTIR, DSC, and TGA measurements were performed to assess the chemical structure and thermal stability of the synthesized hydrogel. Aniline pentamer decreased the swelling capacity, degradation, and drug release rate. Further, contact angle, melting point, and gelation time of hydrogels were enhanced by addition of aniline oligomer. Moreover, it endowed the on-demand electro-responsive drug release. Injectability of hydrogel was evaluated by rheometry, exhibiting proper gelling time at the body temperature. The ionic/electrical conductivity and desired in vitro biocompatibility with PC12 cells were also achieved. Injection of VEGF-loaded electroactive hydrogel in the hippocampal ischemic animal model resulted in decreased infarction volume, improved hippocampal dependent learning, and memory performance. Taken all together, the results confirmed that fabricated injectable hydrogel would be a suitable candidate for ischemic defect treatment and can lead to new horizons to treat neurological disorders.

Zeolites for theranostic applications.

Theranostic platforms bring about a revolution in disease management. During recent years, theranostic nanoparticles have been utilized for imaging and therapy simultaneously. Zeolites, because of their porous structure and tunable properties, which can be modified with various materials, can be used as a delivery agent. The porous structure of a zeolite enables it to be loaded and unloaded with various molecules such as therapeutic agents, photosensitizers, biological macromolecules, MRI contrast agents, radiopharmaceuticals, near-infrared (NIR) fluorophores, and microbubbles. Furthermore, theranostic zeolite nanocarriers can be further modified with targeting ligands, which is highly interesting for targeted cancer therapies.

Poloxamer: A versatile tri-block copolymer for biomedical applications.

Poloxamers, also called Pluronic, belong to a unique class of synthetic tri-block copolymers containing central hydrophobic chains of poly(propylene oxide) sandwiched between two hydrophilic chains of poly(ethylene oxide). Some chemical characteristics of poloxamers such as temperature-dependent self-assembly and thermo-reversible behavior along with biocompatibility and physiochemical properties make poloxamer-based biomaterials promising candidates for biomedical application such as tissue engineering and drug delivery. The microstructure, bioactivity, and mechanical properties of poloxamers can be tailored to mimic the behavior of various types of tissues. Moreover, their amphiphilic nature and the potential to self-assemble into the micelles make them promising drug carriers with the ability to improve the drug availability to make cancer cells more vulnerable to drugs. Poloxamers are also used for the modification of hydrophobic tissue-engineered constructs. This article collects the recent advances in design and application of poloxamer-based biomaterials in tissue engineering, drug/gene delivery, theranostic devices, and bioinks for 3D printing. STATEMENT OF SIGNIFICANCE: Poloxamers, also called Pluronic, belong to a unique class of synthetic tri-block copolymers containing central hydrophobic chains of poly(propylene oxide) sandwiched between two hydrophilic chains of poly(ethylene oxide). The microstructure, bioactivity, and mechanical properties of poloxamers can be tailored to mimic the behavior of various types of tissues. Moreover, their amphiphilic nature and the potential to self-assemble into the micelles make them promising drug carriers with the ability to improve the drug availability to make cancer cells more vulnerable to drugs. However, no reports have systematically reviewed the critical role of poloxamer for biomedical applications. Research on poloxamers is growing today opening new scenarios that expand the potential of these biomaterials from "traditional" treatments to a new era of tissue engineering. To the best of our knowledge, this is the first review article in which such issue is systematically reviewed and critically discussed in the light of the existing literature.

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins.

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.

Intracellular Delivery of Glucose Oxidase for Enhanced Cytotoxicity toward PSMA-Expressing Prostate Cancer Cells.

Reactive oxygen species (ROS) forming enzymes are of significant interest as anticancer agents due to their potent cytotoxicity. A key challenge in their clinical translation is attaining site-specific delivery and minimizing biodistribution to healthy tissues. Here, complexes composed of the ROS enzyme glucose oxidase (GOX), poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG), and anti-prostate specific membrane antigen (anti-PSMA) monoclonal antibody are synthesized for localized delivery and uptake in prostate cancer cells. Formation of anti-PSMA-PLL-g-PEG/GOX results in nanoscale complexes ≈30 nm in diameter with a ζ-potential of 6 mV. The anti-PSMA-PLL-g-PEG/GOX complexes show significant cytotoxicity (≈60% reduction in cell viability) against PSMA-expressing LNCaP cells compared to unmodified GOX. Importantly, cytotoxicity in LNCaP cells occurrs concurrently with anti-PSMA-PLL-g-PEG/GOX uptake and increases in intracellular generation of ROS. These results demonstrate that cytotoxicity of ROS inducing enzymes can be enhanced by intracellular delivery compared to equivalent concentrations of free enzyme, providing a novel means for cancer therapy.

Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat.

We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30-33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers.

Identification of Excipients for Stabilizing Fiberless Adenovirus as Biopharmaceuticals.

Reducing the promiscuous tropism of native adenovirus by using fiberless adenovirus is advantageous toward its use as a gene therapy vector or vaccine component. The removal of the fiber protein on native adenovirus abrogates several undesirable interactions; however, this approach decreases the particle's physical stability. To create stable fiberless adenovirus for pharmaceutical use, the effects of temperature and pH on the particle's stability profile must be addressed. Our results indicate that the stability of fiberless adenovirus is increased when it is stored in mildly acidic conditions around pH 6. The stability of fiberless adenovirus can be further enhanced by using excipients. Excipient screening results indicate that the nonionic surfactant Pluronic F-68 and the amino acid glycine are potential stabilizers because of their ability to increase the thermal transition temperature of the virus particle and promote retention of biological activity after exposure to prolonged thermal stress. Our data indicate that the instability of fiberless adenovirus can be ameliorated by storing the virus in the appropriate environment, and it should be possible to further optimize the virus so that it can be used as a biopharmaceutical.

Nanoparticle Attachment to Erythrocyte Via the Glycophorin A Targeted ERY1 Ligand Enhances Binding without Impacting Cellular Function.

PURPOSE: Nanoparticle (NP) attachment to biocompatible secondary carriers such as red blood cell (RBC) can prolong blood residence time of drug molecules and help create next-generation nanotherapeutics. However, little is known about the impact of RBC-targeted NPs on erythrocyte function. METHODS: The objectives of this study were to develop and characterize in vitro a novel poly-L-lysine (PLL) and polyethylene glycol (PEG) copolymer-based NP containing fluorescent-tagged bovine serum albumin (BSA), and conjugated with ERY1, a 12 amino acid peptide with high affinity for the RBC membrane protein glycophorin A (ENP). RESULTS: Confocal and flow cytometry data suggest that ENPs efficiently and irreversibly bind to RBC, with approximately 70% of erythrocytes bound after 24 h in a physiologic flow loop model compared to 10% binding of NPs without ERY1. Under these conditions, synthesized ENPs were not toxic to the RBCs. The rheological parameters at the applied shear. (0-15 Pa) were not influenced by ENP attachment to the RBCs. However, at high concentration, the strong affinity of ENPs to the glycophorin-A reduced the deformability of the RBC. CONCLUSIONS: ENPs can be efficiently attached to the RBCs without adversely affecting cellular function, and this may potentially enhance circulatory half-life of drug molecules.

Effects of cell-penetrating peptides on transduction efficiency of PEGylated adenovirus.

BACKGROUND: Adenovirus (Ad) is one of the viral vectors most widely used for gene delivery. The virus, however, has serious shortcomings such as immunogenicity, promiscuous tropism, and the inability to efficiently infect certain types of cells. The goal of this study was to improve the ability of an Ad-based vector to efficiently transform cells that lack the native coxsackie-adenovirus receptor (CAR(-)) by modifying the virus with CPP-PEG conjugates. METHODS: The vector was produced by PEGylating Ad, which packages a lacZ reporter gene, and then conjugating CPPs to form CPP-PEG-Ad particles. The study compared the effectiveness of four different CPPs: Pen, Tat, Pep1, and pArg. The effects of CPP amount per virus, degree of PEGylation, and PEG molecular weight on transduction efficiency were studied on CAR(-) NIH/3T3 cells. RESULTS: CPP-PEG-Ad particles transduced CAR(-) cells significantly better than unmodified Ad. Pen, the most effective CPP, produced an 80-fold improvement in transduction compared to the unmodified virus. The Pen peptide utilized a combination of electrostatic and hydrophobic interactions with the cell membrane to maximize cellular association while the other CPPs used only electrostatic or hydrophobic interactions but not both. Lastly, higher degrees of PEGylation, which prompted PEG to adopt a "brush" conformation, resulted in more efficient CPP-PEG-Ad particles because of both better conjugation of CPPs to the PEGylated virus and better exposure of the conjugated CPPs on the surface of the particle. CONCLUSIONS: CPP-PEG-Ad particles efficiently deliver genes to cells that Ad alone would not efficiently infect, thereby extending potential gene therapy treatments to a much broader range of cell types and diseases.

The effect of fiber truncations on the stability of adenovirus type 5.

While fiberless adenovirus has the potential for use as a vaccine or gene delivery vector, some groups have observed instability issues associated with the modified virus. To investigate the effect of fiber modification on adenovirus stability, we produced mutant adenovirus particles that contained the tail and a portion of the shaft domain without the knob. The shaft domain was either completely removed (i.e., fiberless) or truncated to 7-, 14-, or 21-repeats. The mutants were evaluated by biophysical characterization techniques to determine their relative stabilities based on temperature-induced changes to the secondary, tertiary, and quaternary structures of the virus and its constituent proteins. Data acquired using circular dichroism, intrinsic/extrinsic fluorescence, and static/dynamic light scattering were compiled into a comprehensive empirical phase diagram, which showed that native adenovirus was the most stable followed by fiberless adenovirus and then the mutants with truncated fiber protein. In summary, the individual biophysical measurements and the empirical phase diagram showed that providing several repeats of shaft protein negatively impacted the structural stability of the virus more so than completely removing the fiber protein.

Polyethylene glycol-grafted polyethylenimine used to enhance adenovirus gene delivery.

An improved adenoviral-based gene delivery vector was developed by complexing adenovirus (Ad) with a biocompatible, grafted copolymer PEG-g-PEI composed of polyethylene glycol (PEG) and polyethylenimine (PEI). Although an Ad-based gene vector is considered relatively safe, its native tropism, tendency to elicit an immune response, and susceptibility to inactivating antibodies makes the virus less than ideal. The goal of the current study was to determine whether Ad could be complexed with a PEG-g-PEI copolymer that would enable the virus to transduce cells lacking the Ad receptor, while avoiding the issues commonly associated with PEI. A copolymer library was synthesized using 2 kDa PEG and either linear or branched PEI (25 kDa) with a PEG to PEI grafting ratio of 10, 20, or 30. The results of the study indicate that PEG-g-PEI/Ad complexes are indeed able to transduce CAR-negative NIH 3T3 cells. The results also demonstrate that the PEG-g-PEI/Ad complexes are less toxic, less hemolytic, and more appropriately sized than PEI/Ad complexes.

Evaluation of cell-penetrating peptide/adenovirus particles for transduction of CAR-negative cells.

Adenovirus (Ad) is a promising gene therapy vector, and is used currently in more than 23% of clinical gene therapy trials. The viral vector, however, has drawbacks such as immunogenicity, promiscuous tropism, and the inability to infect certain types of cells. The focus of this work was to develop an improved vector through electrostatic formation of a complex between negatively charged Ad and positively charged cell-penetrating peptides (CPPs), including Tat, Penetratin, polyarginine, and Pep1. The resulting complexes were demonstrated to be capable of transducing cells that lack the coxsackie-adenovirus receptor (CAR), and are otherwise difficult to infect with native Ad. The transduction efficiency of the complexes was optimized by varying the multiplicity of infection, complex formation time, and ratio of CPPs to Ad, which improved the transduction efficiency of CPP/Ad on CAR-negative cells more than 100-fold compared with unmodified Ad. The size of the CPP/Ad complex was initially less than 300 nm, but stability studies performed in the presence of serum indicate that the complex aggregates with serum after an extended period of time. The results of the current study indicate that electrostatic modification of Ad with CPPs provides a relevant platform for developing effective Ad-based gene therapy vectors.

Protein impurities from cell culture dramatically impact transduction efficiency of polymer/virus hybrid vectors.

Polyethylenimine (PEI) was used recently with murine leukemia virus-like particles (MLV-VLPs) to produce a hybrid vector that possesses advantages over the native virus; the transduction efficiency of this vector, however, was less than the transduction efficiency of the native virus. The cause of the reduced efficiency was hypothesized to be related to the involvement of proteins in PEI/MLV-VLP complex formation and overall complex size. To test the hypothesis and potentially improve the efficiency of the hybrid vector, ultracentrifugation and size exclusion chromatography were used to purify MLV-VLP and to study the effect of proteins in cell culture medium on complex formation. Based on dynamic light scattering and electron microscopy, complexes formed from the purified MLV-VLPs were smaller, but surprisingly, less efficient than complexes formed from unpurified MLV-VLPs. The addition of protein to purified MLV-VLPs showed that the initial efficiency could be restored and that the purification strategy was not inactivating the MLV-VLPs. Further, by optimizing the amount of protein added to the purified MLV-VLPs, the level of transduction by PEI/MLV-VLP improved 1.6-fold. Particle characterization showed a correlation between the size of the PEI/MLV-VLP complex and the transduction efficiency, which is likely a result of greater sedimentation and cell contact during in vitro studies.

A top-down approach for construction of hybrid polymer-virus gene delivery vectors.

Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells.

Engineering of a stable retroviral gene delivery vector by directed evolution.

The lack of safe and effective delivery vectors continues to be a critical limitation facing human gene therapy. Viruses offer excellent efficiency but can be difficult and expensive to produce and purify. For example, the production and efficiency of murine leukemia virus (MLV) are limited by its inherent instability; the half-life of infectivity is 5-8 hours at 37 degrees C. In order to generate a stable MLV, we randomly mutated the virus genome and selected for infectivity after prolonged incubation at 37 degrees C. After seven rounds of incubation and infection, we isolated a pool of MLV variants with double the half-life of wild-type MLV. Remarkably, a single mutation in the viral protease (PR), G119E, was responsible for the enhanced stability. Saturation mutagenesis at residue 119 revealed variants with half-lives of approximately 24 hours at 37 degrees C. Double mutants combining the changes at position 119 of the PR and substitutions in the PR substrate-binding pocket exhibited half-lives of up to approximately 40 hours. MLV variants provided two- to fourfold higher viral titers and exhibited increased stability with various wild-type envelope proteins. The improved stability of the variant MLVs will provide more facile virus production and increased transduction efficiency.