Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents.

Elastin-like polypeptides (ELP) are a class of materials that are widely used as purification tags and in potential therapeutic applications. We have used the hydrophobic nature of ELP to extract them into organic solvents and precipitate them to obtain highly pure materials. Although many different types of ELP have been rapidly purified in this manner, the underlying mechanism for this process and its ability to retain functional proteins within organic phase-rich media has been unclear. A cleavable ELP-Intein construct fused with the enzyme chorismate mutase (ELP-I-Cm2) was used to better understand the organic solvent extraction process for ELP and the factors impacting the retention of enzyme activity. Our extraction studies indicated that a cell lysis step was essential to stabilize the ELP-I-Cm2 in the organic phase, prevent intein cleavage, and extract the fusion protein with high efficiency and retained activity. Circular dichroism and infrared spectroscopic characterization of ELP-I-Cm2 in organic solvents and aqueous solutions of the extracted and precipitated material indicated that the ELP secondary structure was retained in both environments. Atomic force microscopy and negative stain transmission electron microscopy imaging of ELP-I-Cm2 in organic solvents revealed highly regular circular features that were ∼50 nm in diameter, in contrast to larger (>100 nm) irregular features found in aqueous solutions. Since reverse micelles have often been used in catalytic processes, we evaluated the enzymatic activity of the ELP-I-Cm2 reversed micelles in different organic solvent mixtures and found that Cm2-mediated reactions in organic media were of comparable rate and efficiency to those in aqueous media. Based on these findings, we report an exciting new opportunity for ELP-enzyme fusion applications by exploiting their ability to form catalytically active reverse micelles in organic media.

Development of a Fast Organic Extraction-Precipitation Method for Improved Purification of Elastin-Like Polypeptides That Is Independent of Sequence and Molecular Weight.

Elastin-like polypeptides (ELP), an increasingly popular tag for protein purification, commonly rely upon inverse transition cycling (ITC) to exploit their lower critical solution temperature characteristics for purification. While considerably faster than chromatography, ITC is still time consuming and often fails to remove host cell contaminants to an acceptable level for in vivo experiments. Here, we present a rapid purification workflow for ELP of broadly varying molecular weight and sequence using a polar organic solvent extraction and precipitation strategy. Four different ELP purification methods were directly compared for their ability to remove host cell protein, nucleic acids, and lipopolysaccharide (LPS) contaminants using a model ELP. On the basis of these findings, an optimized extraction-precipitation method was developed that gave highly pure ELP from bacterial pellets in approximately 2.5 h while removing major host cell contaminants, including LPS to levels below 1 EU/mL, to produce highly pure material that is suitable for in vivo applications. Application of this method to the rapid purification of an ELP-epidermal growth factor fusion gave an isolate that retained its capacity to bind to epidermal growth factor receptor positive cells, thereby demonstrating that this method is capable of producing a functional construct after purification by organic extraction-precipitation.

Immunostimulatory Response of RWFV Peptide-Targeted Lipid Nanoparticles on Bladder Tumor Associated Cells.

Bladder carcinoma is the most expensive tumor type to treat on a cost-per-patient basis from diagnosis to death. Treatment with Bacillus Calmette Guerin (BCG) instillation is the only approved immunotherapy in the clinic for the remission of superficial bladder carcinoma. Unfortunately, frequent relapses, high local morbidity, risk of systemic mycobacterial infection, and occasional supply chain interruptions limit the utility of BCG for bladder cancer treatment. It is well known that BCG utilizes an adhesin protein known as fibronectin attachment protein that possesses a crucial RWFV peptide sequence for binding to the bladder tumor microenvironment prior to the initiation of the immunotherapeutic response. We report a RWFV-targeted, pH-responsive stabilized lipid nucleic acid nanoparticle (LNP) vehicle for the effective delivery of an immunotherapeutic oligonucleotide, CpG, that is assembled using a glass microfluidic Chemtrix 3221 reactor. Our small-angle X-ray scattering studies revealed a layer-by-layer assembly of the oligonucleotides with a repeat distance of 6.04 nm within the LNP. Using flow cytometry to evaluate the different cell types found in the bladder tumor microenvironment, RWFV-targeted LNPs were found to attach specifically to fibronectin-secreting cells in culture during a 2 h incubation period. The trafficking and cellular fate of these targeted LNPs were revealed by confocal microscopy of RAW264.7 macrophages to enter the endocytotic pathway within 4 h post treatment. Importantly, control studies reveal that only the pH-sensitive LNP formulation is capable of efficiently releasing the payload within 12 h. As a result, the targeted pH-sensitive LNP resulted in higher expression levels of costimulatory molecules CD83, CD 86, and MHC II, while also inducing higher levels of TNF-α secretion from macrophages. These results demonstrate that RWFV-targeted, pH-sensitive LNP formulations are capable of maximum immunotherapeutic response, potentially making them a highly efficient, lower risk, and readily manufactured alternative to BCG immunotherapy.

Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules.

BACKGROUND: Bladder cancer is the fourth most common cancer in men and eleventh most common in women. Combination therapy using a gene and chemotherapeutic drug is a potentially useful strategy for treating bladder cancer in cases where a synergistic benefit can be achieved successfully. This approach relies on developing drug combinations using carrier systems that can load both hydrophilic genes and hydrophobic drugs. Ideally, the formulation for carrier system should be free of traditional high shear techniques such as sonication and extrusion to reduce shear-induced nucleic acid strand breakage. Moreover, the system should be able to protect the nucleic acid from enzymatic attack and deliver it specifically to the tumor site. MATERIALS AND METHODS: A dual payload carrier system that was formulated using a simple flow mixing technique to complex anionic plasmid (EGFP-NLS) using a cationic polymer (CD-PEI2.5kD) followed by coating of the polyplex using lipid membranes. The resulting lipid-coated polyplex (LCP) formulations are targeted to bladder cancer cells by employing a bacterial adhesive peptide sequence, RWFV, that targets the LCP to the tumor stroma for efficiently delivering reporter plasmid, EGFP-NLS and a model small molecule drug, pyrene, to the cancer cells. RESULTS: Encapsulation efficiency of the peptide targeted carrier for the plasmid was 50% ± 0.4% and for pyrene it was 16% ± 0.4%. The ability of the targeted LCP to transfect murine bladder cancer cells was 4-fold higher than LCP bearing a scrambled peptide sequence. Fluorescence of cells due to pyrene delivery was highest after 4 hrs using targeted LCP. Finally, we loaded the peptide targeted LCP with anti-cancer agent, curcumin. The targeted formulation of curcumin resulted in only 45% viable cancer cells at a concentration of 5 µg/mL, whereas the empty and non-targeted formulations did not result any significant cell death. CONCLUSION: These results demonstrate the specificity of the targeting peptide sequence in engaging tumor cells and the utility of the developed carrier platform to deliver a dual payload to bladder tumor cells.

An Implantable Ultrasonically-Powered Micro-Light-Source (µLight) for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising cancer treatment modality that can selectively target unresectable tumors through optical activation of cytotoxic agents, thus reducing many side effects associated with systemic administration of chemotherapeutic drugs. However, limited light penetration into most biological tissues have so far prevented its widespread adoption beyond dermatology and a few other oncological applications in which a fiber optic can be threaded to the desired locations via an endoscopic approach (e.g., bladder). In this paper, we introduce an ultrasonically powered implantable microlight source, µLight, which enables in-situ localized light delivery to deep-seated solid tumors. Ultrasonic powering allows for small receiver form factor (mm-scale) and power transfer deep into the tissue (several centimeters). The implants consist of piezoelectric transducers measuring 2 × 2 × 2 mm3 and 2 × 4 × 2 mm3 with surface-mounted miniature red and blue LEDs. When energized with 185 mW/cm2 of transmitted acoustic power at 720 kHz, µLight can generate 0.048 to 6.5 mW/cm2 of optical power (depending on size of the piezoelectric element and light wavelength spectrum). This allows powering multiple receivers to a distance of 10 cm at therapeutic light output levels (a delivery of 20-40 J/cm2 light radiation dose in 1-2 hours). In vitro tests show that HeLa cells irradiated with µLights undergo a 70% decrease in average cell viability as compared to the control group. In vivo tests in mice implanted with 4T1-induced tumors (breast cancer) show light delivery capability at therapeutic dose levels. Overall, results indicate implanting multiple µLights and operating them for 1-2 hours can achieve cytotoxicity levels comparable to the clinically reported cases using external light sources.

Gd3+:DOTA-Modified 2-Hydroxypropyl-ß-Cyclodextrin/4-Sulfobutyl Ether-ß-Cyclodextrin-Based Polyrotaxanes as Long Circulating High Relaxivity MRI Contrast Agents.

A family of five water-soluble Gd3+:1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-ß-cyclodextrin and 4-sulfobutylether-ß-cyclodextrin macrocycles threaded onto Pluronic cores were developed as long circulating magnetic resonance contrast agents. Short diethylene glycol diamine spacers were utilized for linking the macrocyclic chelator to the PR scaffold prior to Gd3+ chelation. The PR products were characterized by 1H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation. Nuclear magnetic relaxation dispersion and molar relaxivity measurements at 23 °C revealed that all the PR contrast agents displayed high spin-spin T1 relaxation and spin-lattice T2 relaxation rates relative to a DOTAREM control. When injected at 0.05 mmol Gd/kg body weight in BALB/c mice, the PR contrast agents increased the T1-weighted MR image intensities with longer circulation times in the blood pool than DOTAREM. Excretion of the agents occurred predominantly via the renal or biliary routes depending on the polyrotaxane structure, with the longest circulating L81 Pluronic-based agent showing the highest liver uptake. Proteomic analysis of PR bearing different ß-cyclodextrin moieties indicated that lipoproteins were the predominant component associated with these materials after serum exposure, comprising as much as 40% of the total protein corona. We infer from these findings that Gd(III)-modified PR contrast agents are promising long-circulating candidates for blood pool analysis by MRI.

Development of dihydrochalcone-functionalized gold nanoparticles for augmented antineoplastic activity.

BACKGROUND: Phloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodium-glucose linked transport 2 (SGLT2) inhibitor. While the aglycone metabolite of phloridzin, phloretin, displays a reduced capacity of SGLT2 inhibition, this nutraceutical displays enhanced antineoplastic activity in comparison to phloridzin. PURPOSE: The objective of this study was to develop gold nanoparticle (AuNP) mediated delivery of phloridzin and phloretin and explore their anticancer mechanism through conjugation of the dihydrochalcones and the AuNP cores. METHODS: Phloridzin and phloretin conjugated AuNPs (Phl-AuNP and Pht-AuNP) were synthesized in single-step, rapid, biofriendly processes. The synthesized AuNPs morphology was characterized via transmission electron microscopy and ultraviolet-visible spectroscopy. The presence of phloridzin or phloretin was confirmed using scanning electron microscopy-energy dispersive x-ray spectroscopy. The percentage of organic component (phloridzin/phloretin) onto AuNPs surface was characterized using thermogravimetric analysis. Assessment of the antineoplastic potency of the dihydrochalcones conjugated AuNPs against cancerous cell lines (HeLa) was accomplished through monitoring via flow cytometry. RESULTS: The functionalized AuNPs were synthesized via a single-step method that relied only upon the redox potential of the conjugate itself and required no toxic chemicals. The synthesized Phl-AuNPs were found to be in the size range of 15±5 nm, whereas the Pht-AuNP were found to be 8±3 nm, placing both conjugated AuNPs well within the size range necessary for successful pharmaceutical applications. These assays demonstrate a significant increase in the cancerous cell toxicities as a result of the conjugation of the drugs to AuNPs, as indicated by the 17.45-fold increase in the efficacy of Pht-AuNPs over pure phloretin, and the 4.49-fold increase in efficacy of Phl-AuNP over pure phloridzin. CONCLUSION: We report a simple, biofriendly process using the reducing and capping potential of the dihydrochalcones, phloridzin and phloretin, to synthesize stable AuNPs that have promising futures as potential antineoplastic agents.

Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction.

Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt. Since the underlying basis for the ELP Tt is related to its high overall hydrophobicity, we anticipated that ELP would be excellent candidates for purification by organic extraction. We report the first method for rapidly purifying ELP directly from whole E. coli cells or clarified lysates using pure organic solvents and solvent mixtures, followed by aqueous back extraction. Our results show that small ELP and a large ELP-fusion protein can be isolated in high yield from whole cells or cell lysates with greater than 95% purity in less than 30 min and with very low levels of LPS and DNA contamination.

Targeting and Internalization of Liposomes by Bladder Tumor Cells Using a Fibronectin Attachment Protein-Derived Peptide-Lipopolymer Conjugate.

A synthetic peptidolipopolymer conjugate, incorporated into liposomes to promote specific binding to the fibronectin (FBN) matrix surrounding bladder tumor cells and promote cellular internalization of FBN-integrin complexes, is reported. The peptide promotes association with MB49 mouse model bladder tumor cells in a sequence-specific and concentration-dependent manner, with the maximum cell association occurring at 2 mol % RWFV-PEG2000-DSPE. Double PEGylation of the liposome membrane (i.e., 4 mol % mPEG1000-DSPE + 2 mol % RWFV-PEG2000-DSPE) enhanced binding by >1.6-fold, by improving ligand presentation on the liposome surface. The sequence specificity of the peptide-lipopolymer construct was confirmed by comparing liposomes containing RWFV-PEG2000-DSPE with scrambled and nonpeptidic lipopolymer liposomal formulations. MB49 tumor-bearing mice showed greater mean radiance values for FAP peptide-targeted liposomes in tumor-associated regions of interest than for nontargeted and scrambled peptide liposome formulations. These findings suggest that peptide-modified liposomes may be an attractive vehicle for targeted delivery to bladder tumors in vivo.

PLGA-PEG nano-delivery system for epigenetic therapy.

Efficient delivery of cytidine analogues such as Azacitidine (AZA) into solid tumors constitutes a primary challenge in epigenetic therapies. We developed a di-block nano-vector based on poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) for stabilization of the conjugated AZA under physiological conditions. With equimolar drug content, our nano-conjugate could elicit a better anti-proliferative effect over free drug in breast cancer both in vitro and in vivo, through reactivation of p21 and BRCA1 to restrict cell proliferation. In addition, we applied single-molecule fluorescence tools to characterize the intracellular behavior of the AZA-PLGE-PEG nano-micelles at a finer spatiotemporal resolution. Our results suggest that the nano-micelles could effectively enrich in cancer cells and may not be limited by nucleoside transporters. Afterwards, the internalized nano-micelles exhibit pH-dependent release and resistance to active efflux. Altogether, our work describes a delivery strategy for DNA demethylating agents with nanoscale tunability, providing a cost-effective option for pharmaceutics.

Impact of surfactant treatment of paclitaxel nanocrystals on biodistribution and tumor accumulation in tumor-bearing mice.

We have previously tested paclitaxel nanocrystals (PTX-NCs) in tumor murine models and learned that the nanocrystal formulation could achieve similar and superior anticancer efficacy to the conventional Taxol® formulation, but with significantly reduced side-effects. The nanocrystals were not coated with any surfactants and a majority of the injected dose was taken up by the liver (>40%), while a minimal amount was present in the blood circulation and quickly eliminated. The aim of this work was to treat the surface of PTX-NCs with PEG-based polymers and examine the impact by surface coating on biodistribution, pharmacokinetics, and tumor retention. Testing in tumor-bearing mice showed that PTX-NCs treated with Pluronic® F68 (PEG-PPG-PEG block polymer) significantly enhanced blood circulation of the drug and accumulation in tumor tissue. The absolute amount reaching the tumor, however, was still minimal relative to the dose.

Structure-property relationship for in vitro siRNA delivery performance of cationic 2-hydroxypropyl-ß-cyclodextrin: PEG-PPG-PEG polyrotaxane vectors.

Nanoparticle-mediated siRNA delivery is a promising therapeutic approach, however, the processes required for transport of these materials across the numerous extracellular and intracellular barriers are poorly understood. Efficient delivery of siRNA-containing nanoparticles would ultimately benefit from an improved understanding of how parameters associated with these barriers relate to the physicochemical properties of the nanoparticle vectors. We report the synthesis of three Pluronic(®)-based, cholesterol end-capped cationic polyrotaxanes (PR(+)) threaded with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) for siRNA delivery. The biological data showed that PR(+):siRNA complexes were well tolerated (∼90% cell viability) and produced efficient silencing (>80%) in HeLa-GFP and NIH 3T3-GFP cell lines. We further used a multi-parametric approach to identify relationships between the PR(+) structure, PR(+):siRNA complex physical properties, and biological activity. Small angle X-ray scattering and cryoelectron microscopy studies reveal periodicity and lamellar architectures for PR(+):siRNA complexes, whereas the biological assays, ζ potential measurements, and imaging studies suggest that silencing efficiency is influenced by the effective charge ratio (ρeff), polypropylene oxide (PO) block length, and central PO block coverage (i.e., rigidity) of the PR(+) core. We infer from our findings that more compact PR(+):siRNA nanostructures arising from lower molecular weight, rigid rod-like PR(+) polymer cores produce improved silencing efficiency relative to higher molecular weight, more flexible PR(+) vectors of similar effective charge. This study demonstrates that PR(+):siRNA complex formulations can be produced having higher performance than Lipofectamine(®) 2000, while maintaining good cell viability and siRNA sequence protection in cell culture.

Ultrastructural analysis of 3 hyaluronic acid soft-tissue fillers using scanning electron microscopy.

BACKGROUND: Although hyaluronic acid (HA) specifications such as molecular weight and particle size are fairly well characterized, little information about HA ultrastructural and morphologic characteristics has been reported in clinical literature. OBJECTIVE: To examine uniformity of HA structure, the effects of extrusion, and lidocaine dilution of 3 commercially available HA soft-tissue fillers. MATERIALS AND METHODS: Using scanning electron microscopy and energy-dispersive x-ray analysis, investigators examined the soft-tissue fillers at various magnifications for ultrastructural detail and elemental distributions. RESULTS: All HAs contained oxygen, carbon, and sodium, but with uneven distributions. Irregular particulate matter was present in RES but BEL and JUV were largely particle free. Spacing was more uniform in BEL than JUV and JUV was more uniform than RES. Lidocaine had no apparent effect on morphology; extrusion through a 30-G needle had no effect on ultrastructure. CONCLUSION: Descriptions of the ultrastructural compositions and nature of BEL, JUV, and RES are helpful for matching the areas to be treated with the HA soft-tissue filler architecture. Lidocaine and extrusion through a 30-G needle exerted no influence on HA structure. Belotero Balance shows consistency throughout the syringe and across manufactured lots.

Decitabine nanoconjugate sensitizes human glioblastoma cells to temozolomide.

In this study, we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) based nanoconjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells than that with the free drug. After synthesis, the highly efficient uptake process and intracellular dynamics of this nanoconjugate were monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nanovector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing positive feedback to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to the excellent internalization and endolysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than that of free drug molecules. Hence, the synthesized nanoconjugate and temozolomide could act in synergy to deliver a more potent and long-term antiproliferative effect against malignant GBM cells.

Molecular weight analyses and enzymatic degradation profiles of the soft-tissue fillers Belotero Balance, Restylane, and Juvéderm Ultra.

BACKGROUND: In this study, the authors sought to determine the molecular weight distribution of three hyaluronic acids-Belotero Balance, Restylane, and Juvéderm Ultra-and their rates of degradation following exposure to hyaluronidase. Lot consistency of Belotero Balance also was analyzed. METHODS: Three lots of Belotero Balance were analyzed using liquid chromatography techniques. The product was found to have high-molecular-weight and low-molecular-weight species. One lot of Belotero Balance was compared to one lot each of Juvéderm Ultra and Restylane. Molecular weights of the species were analyzed. The hyaluronic acids were exposed to ovine testicular hyaluronidase at six time points-baseline and 0.5, 1, 2, 6, and 24 hours-to determine degradation rates. RESULTS: Belotero Balance lots were remarkably consistent. Belotero Balance had the largest high-molecular-weight species, followed by Juvéderm Ultra and Restylane (p < 0.001). Low-molecular-weight differences among all three hyaluronic acids were not statistically significant. Percentages of high-molecular-weight polymer differ among the three materials, with Belotero Balance having the highest fraction of high-molecular-weight polymer. Degradation of the high-molecular-weight species over time showed different molecular weights of the high-molecular-weight fraction. Rates of degradation of the hyaluronic acids following exposure to ovine testicular hyaluronidase were similar. All hyaluronic acids were fully degraded at 24 hours. CONCLUSIONS: Fractions of high-molecular-weight polymer differ across the hyaluronic acids tested. The low-molecular-weight differences are not statistically significant. The high-molecular-weight products have different molecular weights at the 0.5- and 2-hour time points when exposed to ovine testicular hyaluronidase and are not statistically different at 24 hours.

Synthesis, characterization, and evaluation of pluronic-based ß-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

Several lines of evidence suggest that ß-cyclodextrin (ß-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current ß-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based ß-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)-polypropylene glycol (PPG)-PEG triblock copolymers. These compounds carry multiple copies of ß-CD as shown by (1)H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free ß-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and (1)H NMR analysis. Filipin staining studies using npc2(-/-) fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric ß-CD alone.

Cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes for siRNA delivery.

RNA interference has broad therapeutic potential due to its high specificity and ability to potentially evade drug resistance. Three cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes derived from polymer axles of different sizes (MW 2,000, 3,400, and 10,000) have been synthesized for delivering siRNA. These polyrotaxanes are able to condense siRNA into positively charged particles that are <200 nm in diameter, enabling their facile internalization into mammalian cells. The cationic polyrotaxanes display cytotoxicity profiles that are >10(2)-fold lower than the commercial standard bPEI and gene silencing efficiencies that are comparable to those of both Lipofectamine 2000 and bPEI. Our findings suggest that the cationic polyrotaxanes display a size-activity relationship, wherein the higher molecular weight polyrotaxanes (PEG3,400 and 10,000) are able to condense and deliver siRNA better than the lower molecular weight material (PEG2,000).

Multi-armed cationic cyclodextrin:poly(ethylene glycol) polyrotaxanes as efficient gene silencing vectors.

A family of branched polyrotaxanes (bPRTx(+)), threaded with multiple cationic α-cyclodextrins (α-CDs) onto a multi-armed poly(ethylene glycol) (PEG) core, were synthesized and studied as gene silencing vectors. These bPRTx(+) formed stable, positively charged complexes with diameters of 150-250 nm at N/P ratios as low as 2.5. The bPRTx(+) materials were shown to have gene-silencing efficiencies comparable to those of Lipofectamine 2000 (L2k) and bPEI, while displaying similar toxicity profiles. The unique structure of these polyrotaxanes allows them to effectively condense and complex siRNA into nanoparticles at much lower N/P ratios than L2k or bPEI. These findings suggest that bPRTx(+) may be useful materials for gene therapy applications.

Acid-labile mPEG-vinyl ether-1,2-dioleylglycerol lipids with tunable pH sensitivity: synthesis and structural effects on hydrolysis rates, DOPE liposome release performance, and pharmacokinetics.

A family of 3-methoxypoly(ethylene glycol)-vinyl ether-1,2-dioleylglycerol (mPEG-VE-DOG) lipopolymer conjugates, designed on the basis of DFT calculations to possess a wide range of proton affinities, was synthesized and tested for their hydrolysis kinetics in neutral and acidic buffers. Extruded ∼100 nm liposomes containing these constructs in ≥90 mol % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) produced dispersions that retained their calcein cargo for more than 2 days at pH 7.5, but released the encapsulated contents over a wide range of time scales as a function of the electronic properties of the vinyl ether linkage, the solution pH, and the mPEG-VE-DOG composition in the membrane. The in vivo performance of two different 90:10 DOPE:mPEG-VE-DOG compositions was also evaluated for blood circulation time and biodistribution in mice, using (125)I-tyraminylinulin as a label. The pharmacokinetic profiles gave a t(1/2) of 7 and 3 h for 90:10 DOPE:ST302 and 90:10 DOPE:ST502, respectively, with the liposomes being cleared predominantly by liver and spleen uptake. The behavior of these DOPE:mPEG-VE-DOG formulations is consistent with their relative rates of vinyl ether hydrolysis, i.e., the more acid-sensitive mPEG-VE-DOG derivatives produced faster leakage rates from DOPE:mPEG-VE-DOG liposomes, but decreased the blood circulation times in mice. These findings suggest that the vinyl ether-based PEG-lipid derivatives are promising agents for stabilizing acid-sensitive DOPE liposomes to produce formulations with a priori control over their pH responsiveness in vitro. Our data also suggest, however, that the same factors that contribute to enhanced acid sensitivity of the DOPE:mPEG-VE-DOG dispersions are also likely responsible for their reduced pharmacokinetic profiles.

DNA-vaccine platform development against H1N1 subtype of swine influenza A viruses.

Swine influenza virus (SIV) is an important viral pathogen in pig populations. However, commercial vaccines cannot provide complete protection with induced humoral immunity only, and require frequent updates to fight against current isolates. DNA vaccination is an effective means of eliciting both arms of the immune system, the humoral and cellular immune responses. In this study, DNA vector pcDNA3.1 was inserted with a chimeric intron downstream of the CMV promoter region followed by a Kozak sequence to enhance the expression of gene inserts. The C-terminal of the VP22 gene (VP22c), encoding the tegument protein of bovine herpesvirus-1, was fused separately to the N-terminal of four quadruplicated epitopes: two B-cell epitopes (HA91-108 and M2e), and two T-cell epitopes (NP366-374 and NP380-393), which were conserved, at least among the three SIV subtypes prevailing in pig populations in North America. Linker -KK- was used to space between each copy of the two B-cell epitopes, and -RVKR- was used for the two T-cell epitopes, in order to enhance the presentation of epitopes to the immune system. The expression of epitopes was confirmed in in vitro transfection of 293FT cells, and higher percentages of epitope-positive cells were achieved from the plasmids containing VP22c than those without. After the DNA plasmids were administered to mice intramuscularly in combination or separately, or boosted with recombinant proteins of quadruplicated epitopes fused to VP22c, the vaccine stimulated the desired epitope-specific humoral immunity to the two B-cell epitopes, and cellular immunity to the epitope NP380-393. Our results indicate that plasmids with quadruplicated epitopes fused to the VP22c may be a potential vehicle in developing epitopes as vaccines against SIV.