DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYMERIC MICELLES FOR GENE-DIRECTED ENZYME PRODRUG THERAPY

Gene-directed enzyme prodrug therapy is a form of cancer therapy in which delivery of a gene that encodes an enzyme is able to convert a prodrug, a pharmacologically inactive molecule, into a potent cytotoxin. Currently delivery of gene and prodrug is a two-step process. Here, we propose a one-step method using polymer nanocarriers to deliver prodrug, gene and cytotoxic drug simultaneously to malignant cells. Prodrugs acyclovir, ganciclovir and 5-doxifluridine were used to directly to initiate ring-opening polymerization of epsilon-caprolactone, forming a hydrophobic prodrug-tagged poly(epsilon-caprolactone) which was further grafted with hydrophilic polymers (methoxy poly(ethylene glycol), chitosan or polyethylenemine) to form amphiphilic copolymers for micelle formation. Successful synthesis of copolymers and micelle formation was confirmed by standard analytical means. Conversion of prodrugs to their cytotoxic forms was analyzed by both two-step and one-step means i.e. by first delivering gene plasmid into cell line HT29 and then challenging the cells with the prodrug-tagged micelle carriers and secondly by complexing gene plasmid onto micelle nanocarriers and delivery gene and prodrug simultaneously to parental HT29 cells. Anticancer effectiveness of prodrug-tagged micelles was further enhanced by encapsulating chemotherapy drugs doxorubicin or SN-38. Viability of colon cancer cell line HT29 was significantly reduced. Furthermore, in an effort to develop a stealth and targeted carrier, CD47-streptavidin fusion protein was attached onto the micelle surface utilizing biotin-streptavidin affinity. CD47, a marker of self on the red blood cell surface, was used for its antiphagocytic efficacy, results showed that micelles bound with CD47 showed antiphagocytic efficacy when exposed to J774A.1 macrophages. Since CD47 is not only an antiphagocytic ligand but also an integrin associated protein, it was used to target integrin alpha(v)beta(3), which is overexpressed on tumor-activated neovascular endothelial cells. Results showed that CD47-tagged micelles had enhanced uptake when treated to PC3 cells which have high expression of alpha(v)beta(3). The synthesized multifunctional polymeric micelle carriers developed could offer a new platform for an innovative cancer therapy regime.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Annexin A5-Conjugated Polymeric Micelles For Dual SPECT and Optical Detection of Apoptosis

Apoptosis, a form of programmed cell death, is critical to homoeostasis, normal development, and physiology. Dysregulation of apoptosis can lead to the accumulation of unwanted cells, such as occurs in cancer, and the removal of needed cells or disorders of normal tissues, such as heart, neurodegenerative, and autoimmune diseases. Noninvasive detection of apoptosis may play an important role in the evaluation of disease states and response to therapeutic intervention for a variety of diseases. It is desirable to have an imaging method to accurately detect and monitor this process in patients. In this study, we developed annexin A5-conjugated polymeric micellar nanoparticles dual-labeled with a near-infrared fluorescence fluorophores (Cy7) and a radioisotope (111In), named as 111In-labeled annexin A5-CCPM. In vitro studies demonstrated that annexin A5-CCPM could strongly and specifically bind to apoptotic cells. In vivo studies showed that apoptotic tissues could be clearly visualized by both single photon emission computed tomography (SPECT) and fluorescence molecular tomography (FMT) after intravenous injection of 111In-labeled Annexin A5-CCPM in 6 different apoptosis models. In contrast, there was little signal in respective healthy tissues. All the biodistribution data confirmed imaging results. Moreover, histological analysis revealed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of apoptosis. In sum, 111In-labeled annexin A5-CCPM allowed visualization of apoptosis by both nuclear and optical imaging techniques. The complementary information acquired with multiple imaging techniques should be advantageous in improving diagnostics and management of patients.

Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

Studies of pseudoachondroplasia chondrocytes and knockdown of the cartilage oligomeric matrix protein

Cartilage oligomeric matrix protein (COMP) is a large, homopentameric, extracellular matrix glycoprotein. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EMD1). These dwarfing conditions are caused by retention of misfolded mutant COMP with type IX collagen and matrilin-3 (MATN3) in the rough endoplasmic reticulum (rER) of the chondrocyte. These proteins form a matrix in the rER that continues to expand until it fills the entire cell, eventually causing cell death. Interestingly, loss of COMP in COMP null mice does not affect normal bone development or growth, suggesting that elimination of COMP (wildtype and mutant) expression may prevent PSACH. The hypothesis of these studies was that a hammerhead ribozyme could eliminate or knockdown COMP mRNA expression in PSACH chondrocytes . To test this hypothesis, a human chondrocyte model system that recapitulates the PSACH chondrocyte phenotype was developed by over-expressing mutant (mt-) COMP in normal chondrocytes using a recombinant adenovirus. Chondrocytes over-expressing mt-COMP developed giant rER cisternae containing COMP, type IX collagen and MATN3. Deconvolution microscopy and computer modeling showed that these proteins formed an ordered matrix surrounding a type II pro-collagen core. Additionally, the results show that a hammerhead ribozyme, ribozyme 56 (Ribo56) reduced over-expressed mt-COMP in COS cells and endogenous COMP in normal chondrocytes and mt-COMP in three PSACH chondrocytes cell line (with different mutations) by 40-70%. Altogether, these studies show that the PSACH cellular phenotype can be created in vitro and that the mt-COMP protein burden can be reduced by the presence of a COMP-specific ribozyme. Future studies will focus on designing ribozymes or short interfering RNA (siRNA) technologies that will result in better knockdown of COMP expression as well as the temporal constraints imposed by the PSACH phenotype. ^

Evidence for phosphorylation and oligomeric assembly of presenilin 1

Pathogenic mutations in presenilin 1 (PS1) are associated with ≈50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12,13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20–23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage λ protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.

Characterization and ageing study of poly(lactic acid) films plasticized with oligomeric lactic acid

Poly(lactic acid) (PLA) was melt-blended with a bio-based oligomeric lactic acid (OLA) plasticizer at different concentrations between 15 wt% and 25 wt% in order to enhance PLA ductility and to get a fully biodegradable material with potential application in films manufacturing. OLA was an efficient plasticizer for PLA, as it caused a significant decrease on glass transition temperature (Tg) while improving considerably ductile properties. Only one Tg value was observed in all cases and no apparent phase separation was detected. Films obtained by compression moulding were stored during 3 months under ambient controlled conditions and thermal, mechanical, structural and oxygen barrier properties were studied in order to evaluate the stability of the PLA–OLA films over time. Blends with 20 and 25 wt% OLA remained stable and compatible with PLA within the ageing period. Besides, PLA–20 wt% OLA formulation was the only one which maintained its amorphous state with adequate thermal, mechanical and oxygen barrier properties for flexible films manufacturing.

Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering

In this study, we investigate the fabrication of 3D porous poly(lactic-co-glycolic acid) (PLGA) scaffolds using the thermally-induced phase separation technique. The current study focuses on the selection of alternative solvents for this process using a number of criteria, including predicted solubility. toxicity, removability and processability. Solvents were removed via either vacuum freeze-drying or leaching, depending on their physical properties. The residual solvent was tested using gas chromatography-mass spectrometry. A large range of porous, highly interconnected scaffold architectures with tunable pore size and alignment was obtained, including combined macro- and microporous structures and an entirely novel 'porous-fibre' structure. The morphological features of the most promising poly(lactic-co-glycolic acid) scaffolds were analysed via scanning electron microscopy and X-ray micro-computed tomography in both two and three dimensions. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.