Electrostatically gated nanofluidic membrane for ultra-low power controlled drug delivery

Patient-centered therapeutic management for chronic medical conditions is a desired but unmet need, largely attributable to the lack of adequate technologies for tailored drug administration. While triggered devices that control the delivery of therapeutics exist, they often rely on impractical continuous external activation. As such, next-generation continuously tunable drug delivery systems independent of sustained external activation remain an elusive goal. Here we present the development and demonstration of a silicon carbide (SiC)-coated nanofluidic membrane that chieves reproducible and tunable control of drug release via electrostatic gating. By applying a low-intensity voltage to a buried electrode, we showed repeatable and reproducible in vitro release modulation of three model analytes. A small fluorophore (Alexa Fluor 647), a large polymer polyIJsodium 4-styrenesulfonate) and a medically relevant agent (DNA), were selected as representatives of small molecule therapeutics, polymeric drug carriers, and biological therapeutics, respectively. Unlike other drug delivery systems, our technology performed consistently over numerous cycles of voltage modulation, for over 11 days. Importantly, low power consumption and minimal leakage currents were achieved during the study. Further, the SiC coating maintained integrity and chemical inertness, shielding the membrane from degradation under simulated physiological and accelerated conditions for over 4 months. Through leveraging the flexibility offered by electrostatic gating control, our technology provides a valuable strategy for tunable delivery, setting the foundation for the next generation of drug delivery systems

Neovascularized implantable cell homing encapsulation platform with tunable local immunosuppressant delivery for allogeneic cell transplantation.

Cell encapsulation is an attractive transplantation strategy to treat endocrine disorders. Transplanted cells offer a dynamic and stimulus-responsive system that secretes therapeutics based on patient need. Despite significant advancements, a challenge in allogeneic cell encapsulation is maintaining sufficient oxygen and nutrient exchange, while providing protection from the host immune system. To this end, we developed a subcutaneously implantable dual-reservoir encapsulation system integrating in situ prevascularization and local immunosuppressant delivery, termed NICHE. NICHE structure is 3D-printed in biocompatible polyamide 2200 and comprises of independent cell and drug reservoirs separated by a nanoporous membrane for sustained local release of immunosuppressant. Here we present the development and characterization of NICHE, as well as efficacy validation for allogeneic cell transplantation in an immunocompetent rat model. We established biocompatibility and mechanical stability of NICHE. Further, NICHE vascularization was achieved with the aid of mesenchymal stem cells. Our study demonstrated sustained local elution of immunosuppressant (CTLA4Ig) into the cell reservoir protected transcutaneously-transplanted allogeneic Leydig cells from host immune destruction during a 31-day study, and reduced systemic drug exposure by 12-fold. In summary, NICHE is the first encapsulation platform achieving both in situ vascularization and immunosuppressant delivery, presenting a viable strategy for allogeneic cell transplantation

Synthesis and Study of Thiophene Containing Thermoshock Protein Hsp90

Darbā ir izdarīts mēģinājums izstrādāt metodes 6-arilmetil-7-alkiltiēno[3,2-d]pirimidīn-4-amīnu un 3,4-asimetriski aizvietotu tiofēnu sintēzei. Ir parādīts, ka vienas no piedāvātām 6-arilmetil-7-alkiltiēno[3,2-d]pirimidīn-4-amīnu sintēzes shēmām no 3,4,5-trimetoksietānskābes praktiska realizācija nav iespējama; otras shēmas – 6-arilmetil-7-alkiltiēno[3,2-d]pirimidīn-4-amīnu sintēze no metil 3-aminotiofēn-2-karboksilāta – praktiska pārbaude šī darba ietvaros netika pabeigta. Ir atrasti apstākļi Suzuki-Mijauras reakcijai, kuros ir izdevies iegūt 3-brom-4-arilaizvietotus tiofēna atvasinājumus no 3,4-dibromtiofēna, kuri tālāk izmantoti 3,4-asimetriski diarilaizvietotu tiofēnu sintēzei. Iespējams, ka atrastos apstākļos var realizēt daudzu līdzīgu 3,4-asimetriski aizvietotu tiofēna atvasinājumu sintēzi.An attempt to elaborate methods of synthesis of 6-arylmethyl-7-alkylthieno[3,2-d]pyrimidin-4-amines and 3,4-unsymmetrically arylsubstituted thiophenes has been undertaken. It was shown, that one of the purposed schemes of synthesis of 6-arylmethyl-7-alkylthieno[3,2-d]pyrimidin-4-amines starting from 3,4,5-trimethoxyphenylacetic acid cannot be practically implemented; the second of the purposed schemes – synthesis of 6-arylmethyl-7-alkylthieno[3,2-d]pyrimidin-4-amines from methyl 3-aminothiophene-2-carboxylate has not been finished. The reaction conditions of Suzuki-Miyaura reaction suitable for the preparation of 3-bromo-4-arylsubstituted thiophenes from 3,4-dibromothiophene, which were then used for further synthesis of 3,4-usymmetrically arylsubstituted thiophene derivatives, have been found

Effects of trehalose polycation end-group functionalization on plasmid DNA uptake and transfection

In this study, we have synthesized six analogs of a trehalose- pentaethylenehexamine glycopolymer (Tr4) that contain (1A) adamantane, (1B) carboxy, (1C) alkynyl-oligoethyleneamine, (1D) azido trehalose, (1E) octyl, or (1F) oligoethyleneamine end groups and evaluated the effects of polymer end group chemistry on the ability of these systems to bind, compact, and deliver pDNA to cultured HeLa cells. The polymers were synthesized in one-pot azide-alkyne cycloaddition reactions with an adaptation of the Carothers equation for step-growth polymerization to produce a series of polymers with similar degrees of polymerization. An excess of end-capping monomer was added at the end of the polymerizations to maximize functionalization efficiency, which was evaluated with GPC, NMR, and MALDI-TOF. The polymers were all found to bind and compact pDNA at similarly low N/P ratios and form polyplexes with plasmid DNA. The effects of the different end group structures were most evident in the polyplex internalization and transfection assays in the presence of serum as determined by flow cytometry and luciferase gene expression, respectively. The Tr4 polymers end-capped with carboxyl groups (1B) (N/P = 7), octyne (1E) (N/P = 7), and oligoethyleneamine (1F) (N/P = 7), were taken into cells as polyplex and exhibited the highest levels of fluorescence, resulting from labeled plasmid. Similarly, the polymers end-functionalized with carboxyl groups (1E at N/P = 7), octyl groups (1E at N/P = 15), and in particular oligoethyleneamine groups (1F at N/P = 15) yielded dramatically higher reporter gene expression in the presence of serum. This study yields insight into how very subtle structural changes in polymer chemistry, such as end groups can yield very significant differences in the biological delivery efficiency and transgene expression of polymers used for pDNA delivery

MAG versus PEG: Incorporating a Poly(MAG) Layer to Promote Colloidal Stability of Nucleic Acid/“Click Cluster” Complexes

Herein, we demonstrate the reversible addition–fragmentation chain transfer (RAFT) synthesis of an adamantane-conjugated glycopolymer, poly­(2-methacrylamido-2-deoxy glucopyranose) (Ad-pMAG), as a hydrophilic coating to promote colloidal stability of click cluster-pDNA complexes in biological media. The Ad-pMAG is assembled via noncovalent interactions through inclusion complex formation between adamantane (Ad) and the β-cyclodextrin (βCD) core of the click cluster/pDNA and then further assembled with plasmid DNA to form polyplexes. Ad-pMAG incorporation was favorable over Ad-poly­(ethylene glycol) (Ad-PEG) due to the enhanced colloidal stability of the click cluster/pDNA polyplex under physiological salt conditions at high N/P ratios. Interestingly, the uptake and reporter gene expression with polyplexes coated with the Ad-pMAG was much lower in HeLa cells than that observed with two glioma cell lines (U87 and U251 cells) in vitro, possibly indicating some delivery specificity

Tunable Thioesters as “Reduction” Responsive Functionality for Traceless Reversible Protein PEGylation

Disulfide has been the only widely used functionality to serve as a reduction responsive trigger in drug delivery. We introduce thioester as a novel thiol responsive chemistry for drug delivery, whose reactivity can be conveniently modulated by choosing the appropriate steric environment around the thioester. Compared with disulfides, thioesters are facile to synthesize and have an order of magnitude broader kinetic tunability. A novel traceless reversible protein PEGylation reagent is developed based on thioester chemistry

Diblock Glycopolymers Promote Colloidal Stability of Polyplexes and Effective pDNA and siRNA Delivery under Physiological Salt and Serum Conditions

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing <i>N</i>-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition–fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000

Glucose-Functionalized, Serum-Stable Polymeric Micelles from the Combination of Anionic and RAFT Polymerizations

Poly­(ethylene-<i>alt</i>-propylene)–poly­[(<i>N</i>,<i>N</i>-dimethylacrylamide)-<i>grad</i>-(2-methacrylamido glucopyranose)] (PEP–poly­(DMA-<i>grad</i>-MAG), or PG) diblock terpolymers were synthesized by combining anionic and reversible addition–fragmentation chain transfer (RAFT) polymerizations. An ω-trithiocarbonate-functionalized PEP homopolymer served as the macromolecular chain transfer agent (macroCTA), and RAFT copolymerizations of DMA and a trimethylsilyl-protected MAG (TMS-MAG) monomer gave a family of PG diblock terpolymers after hydrolysis. The terpolymers had similar degrees of polymerization, and the MAG content ranged from 3.5 to 39 mol % in the hydrophilic block. At 70 °C, the reactivity ratios of DMA (1) and TMS-MAG (2) were determined to be <i>r</i>₁ = 1.86 ± 0.07 and <i>r</i>₂ = 0.16 ± 0.01, and thus the poly­(meth)­acrylamide blocks in the PG diblock terpolymers were likely to be gradient copolymers. Micellar dispersions from PG diblock polymers in water were examined by cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). Spherical micelles with core radii of ca. 7 nm and overall hydrodynamic radii of ca. 15 nm were the predominant morphologies observed in all samples prepared by sequential nanoprecipitation and dialysis. The electron-dense MAG moieties greatly increased the native contrast of the micellar coronae, which were clearly viewed as gray halos around the micellar cores in samples with relatively large MAG content. The stability of the glucose-installed micelles was tested in four biologically relevant media, from simple phosphate-buffered saline (PBS) to fetal bovine serum (FBS), using a combination of DLS and cryo-TEM measurements. Micellar dispersions from a PG diblock terpolymer with 16 mol % of MAG of the hydrophilic block were stable in 100% FBS over at least 14 h, suggesting their minimal interactions with serum proteins. Control experiments suggested that micelles composed of PDMA alone in the corona had similar serum stabilities. These sugar-functionalized micelles hold promise as <i>in vivo</i> drug delivery vehicles to possibly prolong circulation time after intravenous administration

Poly(trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective Polyplex-Mediated siRNA Delivery

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly­(methacrylamidotrehalose) or “poly­(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (<i>n</i> = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P­(AEMA) and P­(AEMA)]. First, we demonstrate that the poly­(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly­(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly­(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly­(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC₅₀ of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly­(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications