Delivery of therapeutics and molecules using self-assembled peptides.

The use of nanobiotechnology in the formulation of drug carriers has been gaining popularity in recent years. Peptide self-assembly technology is a particularly attractive option due to its simplicity and programmability. Selfassembling peptide amphiphiles are surfactant-like molecules that are capable of spontaneous organization into a variety of nanostructures. The structural and functional features of these nanostructures can be designed through alterations to the peptide sequence. With a keen understanding of the supramolecular principles governing the non-covalent interactions involved, drug loading strategies can be customised. Hydrophobic drugs can be hidden within the core via aromatic interactions while gene-based therapeutics can be complexed with a cationic region of lysine residues. This review article focuses on the application of self-assembling peptide amphiphiles to drug delivery in the area of anti-cancer therapeutics, protein- and peptide-based therapeutics and nucleic acid-based therapeutics. Specific examples are used to discuss the various systems available and emphasis is given to the encapsulation and release mechanism.

Thermoresponsive comb-shaped copolymer-Si(100) hybrids for accelerated temperature-dependent cell detachment.

Well-defined comb-shaped copolymer-Si(100) hybrids were prepared, via successive surface-initiated atom transfer radical polymerizations (ATRPs) of glycidyl methacrylate (GMA) and N-isopropylacrylamide (NIPAAm), for accelerated cell detachment at a lower temperature. The Si-C bonded comb copolymer consisted of a well-defined (nearly monodispersed) poly(glycidyl methacrylate) (P(GMA)) main chain, a well-defined NIPAAm polymer (P(NIPAAm) block, and well-defined P(NIPAAm) side chains. The ring opening reaction of epoxy groups of the P(GMA) main chain with 2-chloropropionic acid resulted in the immobilization of the alpha-chloroester groups (the ATRP initiators for the NIPAAm side chains) and the concomitant formation of hydroxyl groups. P(NIPAAm) acted as the thermoresponsive side chains of the comb copolymer for control of cell adhesion and detachment, while the P(GMA) main chain with hydroxyl groups provided a local hydrophilic microenvironment. The unique microstructure of the comb copolymer brushes facilitated cell recovery at 20 degrees C (below the lower critical solution temperature (LCST) of P(NIPAAm)) without restraining cell attachments and growth at 37 degrees C. The accelerated detachment of cells indicated that the underlying hydrophilic environment of the comb copolymer brushes contributed to speedy hydration of the P(NIPAAm) segments below the LCST. The thermoresponsive comb copolymer-Si(100) hybrids are potentially useful as adhesion modifiers for cells in silicon-based biomedical devices.

Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) with varying compositions.

Thermally sensitive block copolymers, poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide)-b-poly(d,l-lactide-co-glycolide) [P(NIPAAm-co-DMAAm)-b-poly(D,L-lactide-co-glycolide) (PLGA)] with different compositions and lengths of PLGA block are synthesized and utilized to fabricate micelles containing doxorubicin (DOX), a model anticancer drug, by a membrane dialysis method for targeted anticancer drug delivery. The critical association concentration (CAC) of the polymers ranges from 4.0 to 25.0 mg/L. An increased length of core-forming block PLGA leads to a decrease in the CAC. The clearly defined core-shell structure of micelles is proved by 1H-NMR analyses of the micelles in CDCl3 and D2O. The morphology of the micelles is analyzed by transmission electron microscopy, showing a spherical structure of both blank and drug-loaded micelles. The results obtained from dynamic light scattering show that the blank and drug-loaded micelles have an average size below 200 nm. The lower critical solution temperature (LCST) of the micelles made from the various polymers is similar, around 39 degrees C in phophate-buffered solution (PBS). The presence of serum in PBS does not alter the LCST significantly. The drug loading capacity varies depending on the PLGA block. The polymers are degradable, and the degradation of PLGA-based polymers is faster than that of poly(lactide) (PLA)-based polymer. The DOX-loaded micelles are stable in PBS containing serum at 37 degrees C but deform at 39.5 degrees C above the normal body temperature, thus triggering DOX release. It is revealed by confocal laser scanning microscopy that free DOX molecules enter cell nuclei very fast and DOX-loaded micelles accumulate mostly in cytoplasm after endocytosis. At a temperature above the LCST, more DOX molecules release from the micelles and enter the nuclei as compared to the temperature below the LCST. DOX-loaded micelles show greater cytotoxicity at a temperature above the LCST. The P(NIPAAm-co-DMAAm)-b-PLGA micelles developed may be a good carrier for anticancer drug delivery.

Thermally sensitive micelles self-assembled from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) for controlled delivery of paclitaxel.

Thermally sensitive micelles self-assembled from poly(N-isopropylacrylamide-co- N,N-dimethylacrylamide)-b-poly(d,l-lactide-co-glycolide)[P(NIPAAm-co-DMAAm)-b-PLGA] are fabricated and used as a carrier for the controlled delivery of paclitaxel. Paclitaxel is efficiently loaded into the micelles by a membrane dialysis method. The lower critical solution temperature (LCST) of the micelles is 39.0 degrees C in PBS. Encapsulation efficiency and loading level of paclitaxel are affected by the initial loading level of paclitaxel, fabrication temperature and polymer composition. The blank and paclitaxel-loaded micelles are characterized by particle size analysis (DLS), morphology (TEM and AFM) and paclitaxel distribution (NMR, DSC and WAXRD). The micelles are spherical in shape, having an average size less than 130 nm. Paclitaxel is molecularly distributed within the core of micelles. Sustained release of paclitaxel is achieved, which is much faster at a temperature above the LCST than at the normal body temperature (37 degrees C). Cytotoxicity of free paclitaxel and paclitaxel-loaded micelles against a human breast carcinoma cell line (MDA-MB-435S) is studied at different temperatures. The cytotoxicity of the paclitaxol-loaded micelles is greater as compared to free paclitaxel. Enhanced cytotoxicity is achieved by the paclitaxol-loaded micelles when the environmental temperature increases slightly above the LCST. Paclitaxel-loaded P(NIPAAm-co-DMAAm)-b-PLGA micelles may provide a good formulation for cancer therapy.

Collagen-coupled poly(2-hydroxyethyl methacrylate)-Si(111) hybrid surfaces for cell immobilization.

To improve the biocompatibility of silicon-based implantable micro- and nanodevices, and to tailor silicon surfaces for controlled cell immobilization, well-defined functional polymer-Si(111) hybrids, consisting of nearly monodispersed poly(2-hydroxyethyl methacrylate [P(HEMA)] with covalently coupled collagen and tethered (Si-C bonded) on the silicon surfaces, were prepared. HEMA was graft polymerized on the hydrogen-terminated Si(111) surface (Si-H surface) via surface-initiated atom transfer radical polymerization (ATRP) to give rise to the Si-g-P(HEMA) hybrid. The active chloride end groups preserved throughout the ATRP process and the chloride groups converted from some (approximately 20%) of the OH groups of the P(HEMA) brushes were used as the leaving groups for nucleophilic reaction with the -NH2 groups of collagen to give rise to the Si-g-P(HEMA)-collagen surface conjugates. These hybrid surfaces were evaluated by culturing 3T3 fibroblasts. The biocompatible Si-g-P(HEMA) hybrid surface resisted attachment and growth of this cell line. The Si-g-P(HEMA)-collagen hybrid surfaces, on the other hand, exhibited good cell adhesion and growth characteristics, and the extent of cell immobilization could be controlled by adjusting the amount of immobilized collagen. Thus, incorporating the collagen-coupled P(HEMA) onto silicon surfaces via robust Si-C bonds may endow the silicon substrates with new and interesting properties for potential applications in silicon-based implantable devices, such as molecular sensors and biochips.

Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers.

Embryonic hippocampal neurons cultured on surface modified fluoropolymers showed enhanced interaction and neurite extension. Poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surfaces were aminated by reaction with a UV-activated mercury ammonia system yielding FEP-[N/O]. Laminin-derived cell-adhesive peptides (YIGSR and IKVAV) were coupled to FEP surface functional groups using tresyl chloride activation. Embryonic (E18) hippocampal neurons were cultured in serum-free medium for up to 1 week on FEP film surfaces that were modified with either one or both of GYIGSR and SIKVAV or GGGGGGYIGSR and compared to control surfaces of FEP-[N/O] and poly(L-lysine)/laminin-coated tissue culture polystyrene. Neuron-surface interactions were analyzed over time in terms of neurite outgrowth (number and length of neurites), cell adhesion and viability. Neurite outgrowth and adhesion were significantly better on peptide-modified surfaces than on either FEP or FEP-[N/O]. Cells on the mixed peptide (GYIGSR/SIKVAV) and the spacer group peptide (GGGGGGYIGSR) surfaces demonstrated similar behavior to those on the positive PLL/laminin control. The specificity of the cell-peptide interaction was demonstrated with a competitive assay where dissociated neurons were incubated in media containing peptides prior to plating. Cell adhesion and neurite outgrowth diminished on all surfaces when hippocampal neurons were pre-incubated with dissolved peptides prior to plating.

Peptide surface modification of poly(tetrafluoroethylene-co-hexafluoropropylene) enhances its interaction with central nervous system neurons.

Poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surfaces were chemically surface modified to introduce one of three laminin adhesive peptides: GYIGSR, GRGDS, or SIKVAV. FEP film surfaces were first reduced with sodium naphthalide to introduce surface carbon-carbon double bonds at two reaction conditions: 20 min at -78 degrees C, and 3 h at 25 degrees C. Scanning electron microscopy and atomic force microscopy indicated that surface topography was unaffected by the reaction conditions. Reduced FEP film surfaces were further modified to introduce hydroxyl groups via hydroboration/oxidation or carboxylic acid groups via oxidation. The hydroxyl (FEP-CHxOH) and carboxylic acid (FEP-COOH) functionalized surfaces provided reactive handles for peptide coupling using tresyl chloride. Surface elemental composition data, determined from X-ray protoelectron spectroscopy, indicated that equivalent amounts of GYIGSR, GRGDS, and SIKVAV were introduced. Two additional coupling reagents, SMCC and TSU, were compared to tresyl chloride for the coupling of radio-labeled tyrosine of GYIGSR. Between 8 and 150 fmol/cm2 of peptide was introduced to the hydroxyl and carboxylic acid functionalized surfaces, with the tresyl coupling reagent showing the greatest amount of peptide incorporated. The tresyl-coupled peptide-modified surfaces were compared in terms of the response of primary, embryonic hippocampal neurons plated from serum-free medium for 4 days. The number and length of neurites extending from the cell bodies were averaged over 50 cells after 1 and 4 days FEP-CHxO-peptide surfaces had either a greater or equivalent hippocampal neuron interaction than the corresponding FEP-COO-peptide surfaces. All peptide-functionalized surfaces had a greater hippocampal neuron interaction than the corresponding FEP-CHxOH, FEP-COOH, and FEP controls after 4 days underlying the importance of the peptides over hydrophilic or hydrophobic surfaces. After 4 days differences in neurite extension were evident among the peptide-functionalized surfaces, with the longest neurites observed on the SIKVAV-functionalized surfaces.

Enhancing the interaction of central nervous system neurons with poly(tetrafluoroethylene-co-hexafluoropropylene) via a novel surface amine-functionalization reaction followed by peptide modification.

Poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) surfaces were modified with cell adhesive peptides, via a novel amination reaction, to enhance the neuron-substrate interaction. Amination of FEP surfaces was achieved by exposing FEP film samples to a UV-activated mercury/ammonia system for either 3 or 24 h, yielding nitrogen compositions of 3.5 and 13.2%, respectively. By labeling the nitrogen functionality with trichlorobenzaldehyde, the surface amine compositions were calculated to be 14 and 4.3% for the 3 and 24 h amination reactions, respectively. Three oligopeptide sequences derived from laminin (GYIGSR, GRGDS, and SIKVAV) were coupled to the aminated FEP (FEP-NH2) surfaces and found to have almost identical surface concentrations as determined by XPS. Using radiolabeled GYIGSR, three coupling agents were compared and the concentration of peptide per surface area was calculated to be 3 and 6 fmol cm-2 for surfaces aminated for 3 and 24 h, respectively, regardless of the coupling agent. The interaction of embryonic hippocampal neurons with the modified surfaces was compared to that with the positive poly(L-lysine)/laminin control in terms of number and length of extended neurites. After 1 day incubation, neurite extension on the GYIGSR- and SIKVAV-coupled surfaces was similar to that on the positive control but significantly greater than that on FEP and FEP-NH2 control surfaces. These peptide-coupled fluoropolymer surfaces enhance the neuron-fluoropolymer interaction, similar to that observed with PLL/laminin.