Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides.

Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.

A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model.

OBJECTIVE: The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening. METHODS: The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting. RESULTS: Pretreatment of cells with MK2i (10 µM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%. CONCLUSION: Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure.

Inhibition of HSP27 phosphorylation by a cell-permeant MAPKAP Kinase 2 inhibitor.

Heat shock protein 27 (HSP27) has been implicated in many intracellular signaling processes. Since the phosphorylation of HSP27 can modulate its activity, the ability to inhibit phosphorylation of HSP27 might have clinical relevance especially with regard to the treatment of fibrosis. We have developed a cell-permeant peptide inhibitor of MAPKAP Kinase 2 (MK2), an enzyme that phosphorylates HSP27, by combining a previously described peptide substrate of MK2 with a cell penetrating peptide. This novel MK2 inhibitor (MK2i) reduced HSP27 phosphorylation by MK2 in vitro. At 10 microM, MK2i inhibited TGF-beta1-induced HSP27 phosphorylation in serum-starved human keloid fibroblasts. In addition, 10 microM MK2i decreased TGF-beta1-induced expression of connective tissue growth factor and collagen type I within serum-starved keloid fibroblasts. Thus, MK2i represents a potential therapeutic for the treatment of fibrotic disorders.

Design of a bioactive cell-penetrating peptide: when a transduction domain does more than transduce.

The discovery of cell-penetrating peptides (CPPs) has facilitated delivery of peptides into cells to affect cellular behavior. Previously, we were successful at developing a phosphopeptide mimetic of the small heat shock-like protein HSP20 . Building on this success we developed a cell-permeant peptide inhibitor of mitogen-activated protein kinase-activated protein kinase 2 (MK2). It is well documented that inhibition of MK2 may be beneficial for a myriad of human diseases including those involving inflammation and fibrosis. During the optimization of the activity and specificity of the MK2 inhibitor (MK2i) we closely examined the effect of cell-penetrating peptide identity. Surprisingly, the identity of the CPP dictated kinase specificity and functional activity to an extent that rivaled that of the therapeutic peptide. The results reported herein have wide implications for delivering therapeutics with CPPs and indicate that judicious choice of CPP is crucial to the ultimate therapeutic success.

Enhanced skin penetration of P20 phosphopeptide using protein transduction domains.

Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent.

Physical matrices stabilized by enzymatically sensitive covalent crosslinks.

This work describes a unique system of gel and gel-like materials formed from physical bonds between heparin and heparin binding peptides (dG-PBD) coupled to multivalent poly(ethylene glycol) vinyl sulfone star polymers (PEGVS) and formed from covalent bonds between an enzymatically sensitive crosslinker and PEGVS. Dynamic mechanical testing revealed that the viscoelastic behavior and gelation kinetics of 10% (w/v) gels formed from 2:1 dG-PBD:PEGVS, heparin, and crosslinker (2:1 gels) and from 3:1 dG-PBD:PEGVS, heparin, and crosslinker (3:1 materials) were significantly influenced by the presence of both physical and covalent bonds. Furthermore, the mechanical properties of both compositions were thermally responsive and reversible. At 4 degrees C, the storage modulus, G', for 2:1 gels (5005.3+/-592.0Pa) and 3:1 materials (5512.0+/-272.7Pa) were statistically similar; however, at 45 degrees C, G' of 2:1 gels decreased to 477.9+/-150.4Pa, and the viscoelastic behavior of 3:1 materials was dominated by viscous behavior. In addition, the mechanical properties of 2:1 gels and 3:1 materials were sensitive to the frequency of the applied stress at 4 degrees C, 20 degrees C, and at 37 degrees C. Although the covalent bonds within the materials provided mechanical stability, the overall viscoelastic response of this system could be dominated by physical crosslinks under certain conditions. Results from degradation studies indicated that the crosslinker was sensitive to collagenase type I but not to thrombin or heparinase I, and a hemolysis assay suggested that the 2:1 gels and 3:1 materials might be biocompatible. These materials could be useful to study the role of physical interactions within networks that mimic the extracellular matrix.

Synthesis and characterization of a poly(lactic-co-glycolic acid) core + poly(N-isopropylacrylamide) shell nanoparticle system.

Poly(lactic-co-glycolic acid) (PLGA) is a popular material used to prepare nanoparticles for drug delivery. However, PLGA nanoparticles lack desirable attributes including active targeting abilities, resistance to aggregation during lyophilization, and the ability to respond to dynamic environmental stimuli. To overcome these issues, we fabricated a nanoparticle consisting of a PLGA core encapsulated within a shell of poly(N-isopropylacrylamide). Dynamic light scattering and transmission electron microscope imaging were used to characterize the nanoparticles, while an MTT assay and ELISA suggested biocompatibility in THP1 cells. Finally, a collagen type II binding assay showed successful modification of these nanoparticles with an active targeting moiety.

Cell-penetrating peptides can confer biological function: regulation of inflammatory cytokines in human monocytes by MK2 inhibitor peptides.

Cell-penetrating peptides have been used as a method of delivering biologically active peptide for over two decades. In this paper, we covalently attached four different cell-penetrating peptides to a peptide that inhibits a kinase important in inflammation, mitogen-activated protein kinase activated protein kinase 2 (MAPKAP2 or MK2). We evaluated the specificity, toxicity, and functionality of these therapeutics in an in vitro model of inflammation using THP-1 monocytes. When treated with the MK2 peptide inhibitors, activated THP-1 human monocytes challenged with lipopolysaccharide (LPS) showed a decrease in TNF-α and IL-6 excretion without apparent toxicity. In addition, western blot analysis revealed decreases in the phosphorylation of heat shock protein 27 (HSP27), a downstream substrate of MK2. These results suggested that our peptides inhibited MK2 activity in vitro and should be investigated further as a potential therapeutic for applications involving inflammation. Furthermore, our results suggested that cell-penetrating peptides can be bioactive.

Tuning cell adhesion by incorporation of charged silicate nanoparticles as cross-linkers to polyethylene oxide.

Controlling cell adhesion on a biomaterial surface is associated with the long-term efficacy of an implanted material. Here we connect the material properties of nanocomposite films made from PEO physically cross-linked with layered silicate nanoparticles (Laponite) to cellular adhesion. Fibroblast cells do not adhere to pure PEO, but they attach to silicate containing nanocomposites. Under aqueous conditions, the films swell and the degree of swelling depends on the nanocomposite composition and film structure. Higher PEO compositions do not support cell proliferation due to little exposed silicate surfaces. Higher silicate compositions do allow significant cell proliferation and spreading. These bio-nanocomposites have potential for the development of biomedical materials that can control cellular adhesion.

Method for tracking nanogel particles in vivo and in vitro.

Hydrogels made of N-isopropylacrylamide (NIPA) can be synthesized in the form of highly monodispersed nanoparticles. After synthesis, NIPA hydrogel nanoparticles (nanogels) can be labeled by Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester through amine-terminated functional groups. This choice of dye is complementary to other biological labeling methods for in vivo studies. When the nanogel/dye nanoparticles are injected into rabbits, they can be imaged via tissue sectioning and confocal microscopy, while nanoparticle concentration can be determined by fluorescent microplate assays. Time-course persistence of nanoparticles in the circulatory system can be readily tracked by direct assay of plasma and urine samples using 485 nm excitation and 538 emission wavelengths to keep background fluorescence to nearly the same level as that found using an empty well. Depending upon how the nanoparticles are injected, circulatory system concentrations can reach high concentrations and diminish to low levels or gradually increase and gradually decrease over time. Injection in the femoral artery results in a rapid spike in circulating nanogel/dye concentration, while injection into the renal artery results in a more gradual increase.

Physical polymer matrices based on affinity interactions between peptides and polysaccharides.

A rapidly forming polymer matrix with affinity-based controlled release properties was developed based upon interactions between heparin-binding peptides and heparin. Dynamic mechanical testing of 10% (w/v) compositions consisting of a 3:1 molar ratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol) to heparin (approximately 18,000 g/mol) revealed a viscoelastic profile similar to that of concentrated, large molecular weight polymer solutions and melts. In addition, the biopolymer mixtures recovered quickly following thermal denaturation and mechanical insult. These gel-like materials were able to sequester exogenous heparin-binding peptides and could release these peptides over several days at rates dependent on relative heparin affinity. The initial release rates ranged from 3.3% per hour for a peptide with low heparin affinity to 0.025% per hour for a peptide with strong heparin affinity. By altering the affinity of peptides to heparin, a series of peptides can be developed to yield a range of release profiles useful for controlled in vivo delivery of therapeutics.