A new sharply pH- and temperature-responsive hydrogel system was designed for delivering drugs to regions of local acidosis, as found in wound healing, tumor sites, or sites of ischemia. The ...reversible addition−fragmentation chain transfer (RAFT) polymerization technique was used to synthesize copolymers of N-isopropylacrylamide (NIPAAM) and propylacrylic acid (PAA) with feed ratios of PAA between 0 and 20 mol %. The pH-responsive viscoelastic properties of these materials as a function of pH and temperature were quantified by rheometry. At physiologic pH (7.4) and 5 wt %, the polymer did not form gels but rather remained soluble at temperatures as high as 50 °C. At lower pH values (pH ca. 5.5 and below), the polymer was liquid at 20 °C, but exhibited a sol−gel phase transformation with increasing temperature and existed as a physical gel at 37 °C. Incorporation of the hydrophobic monomer, butyl acrylate, into the random copolymer raised the pH of gel formation to greater than 6.0 at 37 °C. Drug loading studies demonstrated that p(NIPAAm-co-PAA) hydrogels are able to maintain the bioactivity of basic fibroblast growth factor following storage in hydrogel for 40 h and can provide sustained pH-dependent release of vascular endothelial growth factor over a period of at least three weeks. This hydrogel system will thus gel at controllable acidic pH values upon injection, and is designed to undergo gradual dissolution as it performs its drug delivery function and the ischemic site returns to physiological pH.
Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog ...of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.
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•A specific inhibitor of an EBV prosurvival Bcl-2 protein was computationally designed•The likelihood of designed proteins correctly folding correlates with activity•The designed inhibitor can initiate apoptosis in EBV-infected cancer cells•Intracellular delivery with a micelle carrier suppresses xenograft tumor growth
A novel computationally designed protein, called BINDI, binds to Epstein-Barr virus Bcl-2 protein with picomolar affinity. BINDI initiates apoptosis in EBV-infected cancer lines, can suppress tumor growth, and extends survival in a xenograft disease model of EBV-positive human lymphoma.
Abstract A pH- and temperature-responsive, injectable hydrogel has been designed to take advantage of the acidic microenvironment of ischemic myocardium. This system can improve therapeutic ...angiogenesis methods by providing spatio-temporal control of angiogenic growth factor delivery. The pH- and temperature-responsive random copolymer, poly( N -isopropylacrylamide-co-propylacrylic acid-co-butyl acrylate) ( p NIPAAm-co-PAA-co-BA), was synthesized by reversible addition fragmentation chain transfer polymerization. This polymer was a liquid at pH 7.4 and 37 °C but formed a physical gel at pH 6.8 and 37 °C. Retention of biotinylated basic fibroblast growth factor (bFGF) between 0 and 7 days after injection into infarcted rat myocardium was 10-fold higher with hydrogel delivery versus saline. Following 28 days of treatment in vivo , capillary and arteriolar densities were increased 30–40% by polymer + bFGF treatment versus saline + bFGF or polymer-only controls. Treatment with polymer + bFGF for 28 days resulted in a 2-fold improvement in relative blood flow to the infarct region versus day 0, whereas saline + bFGF or polymer-only had no effect. Fractional shortening determined by echocardiography was significantly higher following treatment with polymer + bFGF (30 ± 1.4%) versus saline (25 ± 1.2%) and polymer alone (25 ± 1.8%). By responding to local changes in pH- and temperature in an animal model of ischemia, this hydrogel system provided sustained, local delivery of bFGF, improved angiogenesis, and achieved therapeutic effects in regional blood flow and cardiac function.
There is a need for simple yet robust biomarker and antigen purification and enrichment strategies that are compatible with current rapid diagnostic modalities. Here, a stimuli-responsive ...nanoparticle system is presented for multiplexed magneto-enrichment and non-instrumented lateral flow strip detection of model antigens from spiked pooled plasma. The integrated reagent system allows purification and enrichment of the gold-labeled biomarker half-sandwich that can be applied directly to lateral flow test strips. A linear diblock copolymer with a thermally responsive poly(N-isopropylacrylamide) (pNIPAm) segment and a gold-binding block composed of NIPAm-co-N,N-dimethylaminoethylacrylamide was prepared by reversible addition–fragmentation chain transfer polymerization. The diblock copolymer was used to functionalize gold nanoparticles (AuNPs), with subsequent bioconjugation to yield thermally responsive pNIPAm-AuNPs that were co-decorated with streptavidin. These AuNPs efficiently complexed biotinylated capture antibody reagents that were bound to picomolar quantities of pan-aldolase and Plasmodium falciparum histidine-rich protein 2 (PfHRP2) in spiked pooled plasma samples. The gold-labeled biomarker half-sandwich was then purified and enriched using 10 nm thermally responsive magnetic nanoparticles that were similarly decorated with pNIPAm. When a thermal stimulus was applied in conjunction with a magnetic field, coaggregation of the AuNP half-sandwiches with the pNIPAm-coated iron oxide nanoparticles created large aggregates that were efficiently magnetophoresed and separated from bulk serum. The purified biomarkers from a spiked pooled plasma sample could be concentrated 50-fold into a small volume and applied directly to a commercial multiplexed lateral flow strip to dramatically improve the signal-to-noise ratio and test sensitivity.
Protein subunit vaccines offer important potential advantages over live vaccine vectors but generally elicit weaker and shorter-lived cellular immune responses. Here we investigate the use of ...pH-responsive, endosomolytic polymer nanoparticles that were originally developed for RNA delivery as vaccine delivery vehicles for enhancing cellular and humoral immune responses. Micellar nanoparticles were assembled from amphiphilic diblock copolymers composed of an ampholytic core-forming block and a redesigned polycationic corona block doped with thiol-reactive pyridyl disulfide groups to enable dual-delivery of antigens and immunostimulatory CpG oligodeoxynucleotide (CpG ODN) adjuvants. Polymers assembled into 23 nm particles with simultaneous packaging of CpG ODN and a thiolated protein antigen, ovalbumin (ova). Conjugation of ova to nanoparticles significantly enhanced antigen cross-presentation in vitro relative to free ova or an unconjugated, physical mixture of the parent compounds. Subcutaneous vaccination of mice with ova–nanoparticle conjugates elicited a significantly higher CD8+ T cell response (0.5% IFN-γ+ of CD8+) compared to mice vaccinated with free ova or a physical mixture of the two components. Significantly, immunization with ova–nanoparticle conjugates electrostatically complexed with CpG ODN (dual-delivery) enhanced CD8+ T cell responses (3.4% IFN-γ+ of CD8+) 7-, 18-, and 8-fold relative to immunization with conjugates, ova administered with free CpG, or a formulation containing free ova and CpG complexed to micelles, respectively. Similarly, dual-delivery carriers significantly increased CD4+IFN-γ+ (Th1) responses and elicited a balanced IgG1/IgG2c antibody response. Intradermal administration further augmented cellular immune responses, with dual-delivery carriers inducing ∼7% antigen-specific CD8+ T cells. This work demonstrates the ability of pH-responsive, endosomolytic nanoparticles to actively promote antigen cross-presentation and augment cellular and humoral immune responses via dual-delivery of protein antigens and CpG ODN. Hence, pH-responsive polymeric nanoparticles offer promise as a delivery platform for protein subunit vaccines.
The development of drug delivery systems based on well-defined polymer nanostructures could lead to significant improvements in the treatment of cancer. The design of these therapeutic nanosystems ...must account for numerous systemic and circulation obstacles as well as the specific pathophysiology of the tumor. Nanoparticle size and surface charge must also be carefully selected in order to maintain long circulation times, allow tumor penetration, and avoid clearance by the reticuloendothelial system (RES). Targeting ligands such as vitamins, peptides, and antibodies can improve the accumulation of nanoparticle-based therapies in tumor tissue but must be optimized to allow for intratumoral penetration. In this review, we will highlight factors influencing the design of nanoparticle therapies as well as the development of modern controlled “living” polymerization techniques (e.g. ATRP, RAFT, ROMP) that are leading to the creation of sophisticated new polymer architectures with discrete spatially-defined functional modules. These innovative materials (e.g. star polymers, polymer brushes, macrocyclic polymers, and hyperbranched polymers) combine many of the desirable properties of traditional nanoparticle therapies while substantially reducing or eliminating the need for complex formulations.
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Design and development of polymers for gene delivery Pack, Daniel W; Hoffman, Allan S; Pun, Suzie ...
Nature reviews. Drug discovery,
200507, 2005-Jul, 2005-07-01, 20050701, Letnik:
4, Številka:
7
Journal Article
Recenzirano
Odprti dostop
The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess ...the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.
Abstract Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation ...chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86–216 nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies.
Traumatic brain injury (TBI) is the leading cause of death and disability in children and young adults, yet there are currently no treatments available that prevent the secondary spread of damage ...beyond the initial insult. The chronic progression of this secondary injury is in part caused by the release of reactive oxygen species (ROS) into surrounding normal brain. Thus, treatments that can enter the brain and reduce the spread of ROS should improve outcome from TBI. Here a highly versatile, reproducible, and scalable method to synthesize core-cross-linked nanoparticles (NPs) from polysorbate 80 (PS80) using a combination of thiol–ene and thiol–Michael chemistry is described. The resultant NPs consist of a ROS-reactive thioether cross-linked core stabilized in aqueous solution by hydroxy-functional oligoethylene oxide segments. These NPs show narrow molecular weight distributions and have a high proportion of thioether units that reduce local levels of ROS. In a controlled cortical impact mouse model of TBI, the NPs are able to rapidly accumulate and be retained in damaged brain as visualized through fluorescence imaging, reduce neuroinflammation and the secondary spread of injury as determined through magnetic resonance imaging and histopathology, and improve functional outcome as determined through behavioral analyses. Our findings provide strong evidence that these NPs may, upon further development and testing, provide a useful strategy to help improve the outcome of patients following a TBI.
Temperature- and pH-sensitive random copolymers of N-isopropylacrylamide (NIPAAm) and propylacrylic acid (PAA) were prepared using the reversible addition fragmentation chain transfer (RAFT) ...polymerization method. The lower critical solution temperatures (LCSTs) (or phase separation temperatures) of the NIPAAm-co-PAA copolymer solutions were measured by the cloud-point method. At slightly acidic conditions, the LCST decreased with increase in PAA content, which suggests that the hydrophobic propyl group of PAA has a greater influence on the LCST than the polar carboxylic acid group at those conditions. An increase of pH led to a significant increase in LCST of the copolymers due to the ionization of the −COOH group. The LCSTs were studied as a function of copolymer composition over the pH range from 5.0 to 7.0. Because the pK a of the polymers can be tuned to fall close to neutral pH, these polymer compositions can be designed to have phase transitions triggered near physiological pH or at slightly acidic pH values that fall within acidic gradients found in biology. The NIPAAm-co-PAA copolymers thus display tunable properties that could make them useful in a variety of molecular switching and drug delivery applications where responses to small pH changes are relevant.
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