abstract: Cancer diseases are among the leading cause of death in the United States. Advanced cancer diseases are characterized by genetic defects resulting in uncontrollable cell growth. Currently, chemotherapeutics are one of the mainstream treatments administered to cancer patients but are less effective if administered in the later stages of metastasis, and can result in unwanted side effects and broad toxicities. Therefore, current efforts have explored gene therapy as an alternative strategy to correct the genetic defects associated with cancer diseases, by administering genes which encode for proteins that result in cell death. While the use of viral vectors shows high level expression of the delivered transgene, the potential for insertion mutagenesis and activation of immune responses raise concern in clinical applications. Non-viral vectors, including cationic lipids and polymers, have been explored as potentially safer alternatives to viral delivery systems. These systems are advantageous for transgene delivery due to ease of synthesis, scale up, versatility, and in some cases due to their biodegradability and biocompatibility. However, low efficacies for transgene expression and high cytotoxicities limit the practical use of these polymers. In this work, a small library of twenty-one cationic polymers was synthesized following a ring opening polymerization of diglycidyl ethers (epoxides) by polyamines. The polymers were screened in parallel and transfection efficacies of individual polymers were compared to those of polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Seven lead polymers that demonstrated higher transgene expression efficacies than PEI in pancreatic and prostate cancer cells lines were identified from the screening. A second related effort involved the generation of polymer-antibody conjugates in order to facilitate targeting of delivered plasmid DNA selectively to cancer cells. Future work with the novel lead polymers and polymer-antibody conjugates developed in this research will involve an investigation into the delivery of transgenes encoding for apoptosis-inducing proteins both in vitro and in vivo. / Dissertation/Thesis / M.S. Chemical Engineering 2011

Dendrimers are a class of polymers with a highly branched, three-dimensional architecture comprised of an initiator core, several interior layers of repeating units, and multiple active surface terminal groups. Dendrimers have been recognized as the most versatile compositionally and structurally controlled nanoscale building blocks for drug and gene delivery. Polyamidoamine (PAMAM) dendrimers have been most investigated because of their unique structures and properties. Polycationic PAMAM dendrimers form compacted polyplexes with nucleic acids at physiological pH, holding great potential for gene delivery. Folate receptor (FRα) is expressed at very low levels in normal tissues but expressed at high levels in cancers in order to meet the folate demand of rapidly dividing cells under low folate conditions. Our primary aim was to investigate folic acid (FA)-conjugated PAMAM dendrimer generation 4 (G4) conjugates (G4-FA) for targeted gene delivery. The in vitro cellular uptake and transfection efficiency of G4-FA conjugates and G4-FA/DNA polyplexes were investigated in Chapter 4. It was found the cellular uptake of G4-FA conjugates and G4-FA/DNA polyplexes was in a FR-dependent manner. Free FA competitively inhibited the cellular uptake of G4-FA conjugates and G4-FA/DNA polyplexes. G4-FA/DNA polyplexes were preferentially taken up by FR-positive HN12 cells but not FR-negative U87 cells. In contrast, the cellular uptake of G4 dendrimers and G4/DNA polyplexes was non-selective via absorptive endocytosis. G4-FA conjugates significantly enhanced cytocompatibility and transfection efficiency compared to G4 dendrimers. This work demonstrates that G4-FA conjugates allow FR-targeted gene delivery, reduce cytotoxicity, and enhance gene transfection efficiency. The in vivo biodistribution of G4-FA conjugates and anticancer efficacy of G4-FA/siRNA polyplexes were investigated in Chapter 5. Vascular endothelial growth factor A (VEGFA) is one of the major regulators of angiogenesis, essential for the tumor development. It was found G4-FA/siVEGFA polyplexes significantly knocked down VEGFA mRNA expression and protein release in HN12 cells. In the HN12 tumor-bearing nude mice, G4-FA conjugates were preferentially taken up by the tumor and retained in the tumor for at least 21 days following intratumoral (i.t.) administration. Two-dose i.t. administration of G4-FA/siVEGFA polyplexes significantly inhibited tumor growth by lowering tumor angiogenesis. In contrast, two-dose i.t. administration of G4/siVEGFA polyplexes caused severe skin lesion, presumably as a result of local toxicity. Taken together, this work shows great potential for the use of G4-FA conjugates in targeted gene delivery and cancer gene therapy. We also explored polyanionic PAMAM dendrimer G4.5 as the underlying carrier to carry camptothecin (CPT) for glioblastoma multiforme therapyin Chapter 6. "Click" chemistry was applied to improve polymer-drug coupling reaction efficiency. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 μM, a 185-fold increase relative to free CPT, presumably as a result of slow release. The conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. This work indicates highly efficient "click" chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery. Immobilizing PAMAM dendrimers to the cell surface may represent an innovative method of enhancing cell surface loading capacity to deliver therapeutic and imaging agents. In Chapter 7, macrophage RAW264.7 (RAW) was hybridized with PAMAM dendrimer G4.0 (DEN) on the basis of bioorthogonal chemistry. Efficient and selective cell surface immobilization of dendrimers was confirmed by confocal microscopy. It was found the viability and motility of RAW-DEN hybrids remained the same as untreated RAW cells. Furthermore, azido sugar and dendrimer treatment showed no effect on intracellular AKT, p38, and NFκB (p65) signaling, indicating that the hybridization process neither induced cell stress response nor altered normal signaling. This work shows the feasibility of applying bioorthogonal chemistry to create cell-nanoparticle hybrids and demonstrates the noninvasiveness of this cell surface engineering approach. In summary, these studies indicate surface-modification of PAMAM dendrimer G4 with FA can effectively target at FR-positive cells and subsequently enhance in vitro transfection efficiency and in vivo gene delivery. G4-FA conjugates may serve as a versatile targeted gene delivery carrier potentially for cancer gene therapy. PAMAM dendrimers G4.5 may serve as a drug delivery carrier for the controlled release of chemotherapeutics. The immune cell-dendrimer hybrids via bioorthogonal chemistry may serve as an innovative drug and gene delivery carrier potentially for cancer chemotherapy. Taken together, engineering of PAMAM dendrimers may advance anticancer drug and gene delivery.

Gene delivery can potentially treat acquired and genetic diseases such as cystic fibrosis, haemophilia and cancer. Non-viral gene delivery vectors are attractive candidates over viral vectors such as recombinant viruses, due to their lower cytotoxicity and immunogenicity, despite significantly lower transfection efficiencies. To improve efficiency of non-viral vectors, the investigation of the various parameters influencing DNA transfection is essential. The present study developed a versatile gene delivery system with tailored physicochemical and biological properties. The system used polymer brushes synthesised via atomic transfer radical polymerisation (ATRP), grafted from silica nanoparticles, whose charge density, grafting density, chemistry, length of brush, the size and shape can be altered. The primary focus of the study was poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), known for its positive charge and DNA condensation. The ability of PDMAEMA to interact with DNA was characterised using dynamic light scattering, electrophoretic light scattering methods, surface plasmon resonance and in situ ellipsometry whilst its interaction with cells was studied via cell viability assays. The brush behaviour in response to pH and ionic strength was also studied. The charge density was altered by copolymerising with poly[oligo(ethylene glycol) methyl ether methacrylate](POEGMA) and the effect of such modification on DNA interaction was studied. PDMAEMA-grafted nanoparticles gave the highest transfection efficiency compared to other synthesised polymer brushes, but still displaying almost 2-fold lower transfection efficiency than the commercially available reagent jetPEI®. Different brush chemistries were also investigated. Poly(glycidyl methacrylate) (PGMA) decorated with oligoamines: allylamine, diethylenetriamine and pentaethylene hexamine, and PDMAEMA quaternized with alkyl halides: methyl iodide, allyl iodide and ethyl iodoacetate did not show any significant transfection, despite their performance reported in the literature. The robust system developed is a promising platform for further investigation of parameters influencing cellular uptake and gene expression, and important milestone to develop non-viral gene delivery systems.

Polymer brushes generated via "grafting-from" approach emerged as an attractive surface modification tool offering chemical stability, synthetic flexibility and unprecedented control over the polymer grafting density, thickness, chemical composition and functionality. They display interesting features to many applications in regenerative medicine including cell culture, tissue engineering and as delivery systems due to exquisite control of physicochemical and biological properties. Cationic polymer brushes are particularly attractive in the field of designing effective vectors for gene delivery as polymer brush allows the design and coating of a variety of particles with well-defined core-shell architecture and chemistry to efficiently condense and deliver nucleic acids. This thesis concentrates on designing safe and efficient gene delivery vectors based on 'graft from' cationic polymer brush and understanding the interaction of nucleic acids with polymer brush. Chapter one presented fundamental knowledge of polymer brush and its biomedical application. The first part of this chapter describes the definition of polymer brush, the preparation strategies, mechanism of atom transfer radical polymerisation and the responsiveness of polymer brush including solvent, pH and ionic strength. The second part discusses the state-of-art applications of polymer brush in regenerative medicine including protein resistant polymer brush for tissue engineering and as drug/gene delivery systems.

No description available.

A major challenge in drug development is ensuring that each new candidate drug is delivered to the appropriate location, in a timely manner and at an optimal concentration. Low drug solubility, drug instability, drug degradation, drug toxicity, or rapid clearance from the body can reduce the effectiveness of an otherwise promising drug candidate. Formulations such as nano/microparticles and melt extruded pellets made with synthetic and natural polymers are effective solutions for the advancement of drug delivery technology. These polymeric formulations can provide controlled release of therapeutic agents by delivering constant doses over long periods, cyclic dosages, and tunable release of both hydrophilic and hydrophobic drugs in order to improve the bioavailability and bioactivity of a drug. PLGA-based nanoparticles formed by emulsion or nanoprecipitation techniques can be designed to have a range of degradation times. Particle degradation and drug release kinetics can be controlled by the physiochemical properties of the polymer, such as molecular weight, hydrophobicity, and polydispersity. This study is focused on developing polymeric-based delivery systems for small and large molecules as treatment strategies for arthrofibrosis and bone tissue engineering. In developing arthrofibrotic treatments, several mechanosignaling and biochemical pathways were targeted using small molecule therapeutics such as blebbistatin (a myosin II ATPase inhibitor), paclitaxel (a microtubule stabilizer), sulfasalazine (a kappa B suppressor), beta-aminopropionitrile (a lysyl oxidase inhibitor) and cis-hydroxyproline (inhibits the formation of stable triple helix structure of collagen). The aforementioned drugs were delivered either via PLGA micro/nanoparticles or via pellets formed by melt extrusion. From the studies performed, it was found that blebbistatin delivered by PLGA nanoparticles could reversibly inhibit fibroblast contractile activity and could significantly inhibit collagen synthesis. These findings lay the foundations for further optimization of drug dosing and potentially enabling a new drug delivery technology for treating arthrofibrosis. Sulfasalazine delivered by melt extruded PLGA pellets significantly inhibited myofibroblast numbers as deduced from α-SMA expression and col1A1 gene expression results and thus can be considered a potential treatment for arthrofibrosis. For bone tissue engineering, plasmids encoding differentiation promoting factors or growth factors such as BMP-2 (pBMP-2), FGF-2 (pFGF-2), PDGF (pPDGF) and VEGF (pVEGF) were delivered via polyethylenimine (PEI), a cationic carrier that interacts electrostatically with negatively charged DNA. The formed nanoplexes were either tested directly or by coating them onto biocompatible titanium metal implants and cultured with human bone marrow derived mesenchymal stem cells (hBMSCs). We found that the combinatorial delivery of pBMP-2 and pFGF-2 significantly enhanced bone regeneration as deduced from Runx-2, alkaline phosphatase and osteocalcin gene expression results as well as from data yielded from alizarin red staining assays and atomic absorption spectroscopy where calcium ion levels were measured. It was also found that pBMP-2 nanoplex-coated titanium discs could significantly enhance bone regenerative gene expression for osteocalcin, Runx-2, and alkaline phosphatase as well as enhance calcium ion expression in human adipose derived mesenchymal stem cells (hADMSCs). Thus, it can be concluded that pFGF-2 and pBMP-2 nanoplexes have osteogenic potential and our studies demonstrate a new methodology with the potential to modify titanium disc implant surfaces for the purposes of enhancing osseointegration.

Drug-resistant pathogens are emerging rapidly and thwart the treatment of common bacterial infectious diseases that can lead to death. Many contagious diseases remain difficult to treat because of acquired drug resistance. Compared to small antibiotics, which interrupt the intracellular biochemical processes, antimicrobial polymers with relatively high molecular weights offer a promising strategy to overcome drug resistance by disrupting the physical integrity of the membrane. Because of the unique mechanism, bacteria need a much longer time to develop resistance. A new class of antimicrobial polymer in which the positive charge and hydrophobic/hydrophilic units are linearly connected in the amidinourea backbone was designed, synthesized, and tested for various bacteria including methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the effects of hydrophobicity and polymer molecular weights on antimicrobial activity by measuring minimum inhibitory concentrations (MIC) and hemolytic activities (HC50). Amidinourea antimicrobial polymers exhibit a promising MIC90 value (13 μg/mL) with low HC50, resulting in high selectivity (HC50/MIC90) against MRSA. Many bacteria have developed resistance against Ciprofloxacin. To overcome the antibiotic resistance associated with Ciprofloxacin, we hypothesized that a steady release of Ciprofloxacin at the bacteria membrane can overcome the drug resistance because the local drug concentration can be overwhelmingly high to suppress the drug efflux pump expressed on the membrane. A series of homo and di-block copolymers containing Ciprofloxacin, as the form of prodrugs, was synthesized using ring-opening metathesis polymerization (ROMP), and we evaluated their antimicrobial efficacy.While homo polymers only containing Ciprofloxacin were inactive against almost all bacteria tested, di-block copolymers containing Cipro and triphenylphosphine exhibited some antimicrobial activity against wild type M. smegmatis. Modulation of chemical environments at the positively charged polymeric materials can significantly influence the biophysical properties required for efficient cellular interaction and subsequent entry. Using intrinsic fluorescent conjugated polymers (CPs), we have demonstrated that the modulated guanidine group with various hydrophilic or hydrophobic moieties dramatically changed their cellular behaviors. We prepared a series of modified guanidine-containing CPs and examined their cellular behaviors by using confocal microscopic imaging. Details of the modification chemistry and modification-dependent cellular behaviors and a knockdown of a target protein in primary cells were discussed.

Prostate cancer is the most common non-skin cancer in America, and the most commonly diagnosed cancer among males. When metastatic, the disease can ultimately be incurable. Consequently, alternative strategies to current treatments are sought, especially in the area of immunotherapy. Vaccine immunotherapy using a specific antigen, such as prostate specific antigen (PSA) seeks to stimulate both the innate and adaptive immune system to destroy tumor cells in the body. PSA is an ideal target antigen given that it has a narrow distribution in tissues and is expressed in virtually all prostate cancer cases. An adenovirus encoding for PSA (Ad-PSA) can be used to deliver the genomic data encoding for PSA production and secretion to the target cell. This type of viral gene delivery system has already been shown to have the potential to stimulate anti-tumor activity. To enhance this activity and increase transfection efficiency, we proposed the combination of a viral system with a non-viral system, in the form of a cationic polymer such as poly(ethyl)enimine (PEI) or chitosan. Cationic polymers complex with the negatively charged adenovirus to form nanoparticles that can be used in gene delivery. Delivery in nanoparticle form can give enhanced uptake by the antigen-presenting cells necessary to initiate the targeted immune response. To further augment this response, previous research has shown that CpG sequences act as an adjuvant to enhance the efficacy of the Ad-PSA vaccines' tumor protection. CpG delivered in particulate form has also been shown to be more effective than delivery in solution. The objective of this proposal was to test the hypothesis that co-delivery of this targeted viral/non-viral gene delivery system will enhance tumor protection in a mouse model of prostate cancer. Using the OVA model antigen system, we found that the adenovirus encoding OVA (AdOVA), coupled with the polymer PEI, enhanced tumor protection in vivo compared to AdOVA alone. To move towards our therapeutic model, these experiments were repeated using chitosan as the cationic polymer carrier, delivering AdOVA, and incorporating CpG into some particles. In this set of experiments, we found that AdOVA + CpG gave the best tumor protection in challenge studies. AdOVA + chitosan + CpG showed a decrease in protective levels and numbers of antigen-specific immune cells. Further experiments focused on elucidating the mechanisms by which chitosan and CpG modulate the immune response. Using the therapeutic AdPSA model, chitosan was not found to enhance tumor protection or numbers of antigen-specific immune cells. Additional experiments found that this depression was not due to problems with viral infectivity or secretion due to chitosan complexation. A series of kinetics studies were performed which showed that peak levels of effector T cells were present 14 days later in AdPSA + CpG immunized mice than in AdPSA alone. This delayed effect may explain the increased levels of protection in AdPSA + CpG mice, and be useful in future vaccine design concerning the timing of peak response.

Gene therapy can potentially treat a wide range of diseases ranging from inherited diseases to cancer. The successful use of nucleic acids to treat genetic diseases is limited by rapid capture and degradation of the nanoparticle by Kupffer cells in the liver. Scavenger receptors on the cell surface, capture both viral and non-viral nanoparticles leading to reduced efficacy. PEG-peptides were found to inhibit scavenger receptors on the surface of Kupffer cells by forming albumin nanoparticles when intravenously dosed. This work explores the development of potent, low-molecular weight PEG-peptide inhibitors. In order to study the in vivo activity of the nanoparticle, an in vivo assay was developed to directly assess the potency of inhibition. High molecular weight polylysine peptides (33.5 kDa) inhibited liver uptake with an IC50 of 18 μM. Incorporation of four leucine residues, to improve albumin binding, allowed for a decrease in PEG molecular weight and number of lysine residues, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys (7.4 kDa) that inhibited scavenger receptors with an IC50 = 20 μM. Further decrease in the PEG molecular weight resulted in the discovery of PEG2kDa- Cys-Tyr- (Leu-Lys4)3-Leu-Lys (4.4 kDa) with potency of 3 μM. The increase in potency could be attributed to a decrease in the zeta potential of the albumin nanoparticle resulting in more efficient scavenger receptor mediated uptake. Co- administration of PEG2kDa- Cys-Tyr-(Leu-Lys4)3-Leu-Lys with a stable PEGylated polyacridine DNA polyplex resulted in inhibition of rapid polyplex uptake by the liver with an IC50 = 11 μM. Other properties including spatial distribution of leucine, hydrophobicity and peptide length were also explored to determine their effect on liver uptake. Hydrophobic peptides resulted in the formation of micelles which were inactive as scavenger receptor inhibitors and exhibited increased liver uptake upon dose escalation. Reduction in the peptide length resulted in peptides that were not captured by the liver. Inhibition scavenger receptors has the potential to improve the efficacy of viral and non-viral nanoparticles. The findings of this work provide a framework for the development of PEG-peptide inhibitors capable of blocking live uptake of viral and non-viral nanoparticles.

Diseases of the liver have a large impact on human health. Genetic disorders, metabolic disorders, alcoholism, cancer, or infections can all impair liver function. If serious enough, a liver transplant may be necessary, a major surgical procedure which requires life-long immune suppression and relies on the availability of donor livers. Gene therapy is being intensively studied as a potential method to treat many disorders, including disorders of the liver. While viral gene therapy has seen some success, possible side effects make it risky, so nonviral gene delivery vectors are being developed. Unfortunately, these nonviral vectors do not yet have the efficiency of the viral vectors. Nonviral gene delivery vectors face many chges in vivo. The vectors must protect DNA from nucleases while it moves through the bloodstream, they must avoid nonspecific uptake, they must be enter the correct cells, and must enter the nucleus before the DNA can be expressed. If any step of this process fails, there will be very little, if any, expression, and it may be impossible to determine what went wrong. One impediment to nonviral gene delivery research is the transition from in vitro studies to in vivo studies. The cancer derived cell lines most often used for in vitro transfections are rapidly dividing, which makes nuclear entry much easier than in the whole animal. While primary cells would be a more accurate model of the in vivo environment, the number of cells that can be obtained from tissues is small, and primary cells usually cannot be cultured for long. This limits the number of experiments that can be done with each preparation of cells. To overcome this, we have miniaturized transfection assays, including the transfection of mouse primary hepatocytes with luciferase in 384 well plates. Because fewer cells are needed, more experiments can be performed with each liver preparation. Another issue introduced by the differences between in vitro and in vivo research is circulatory stability. In vitro, large particles with strong positive charges are desired, because they sink down onto the cells and are attracted to the negatively charged cellular membranes. However, in vivo these particles will aggregate serum proteins and become lodged in narrow capillary beds in the lungs or other organs, often causing toxicity. While this behavior can usually be overcome through PEGylation, improving a particle's circulatory half-life will still improve its chances of finding the correct target. Scavenger receptors found on liver nonparenchymal cells are very efficient at removing negatively charged particles from the bloodstream. We have shown that dosing large amounts of PEGylated polyacridine DNA polyplex can saturate the scavenger receptors and improve circulatory half-life. We have also shown that large doses of PEGylated peptide with or without acridine groups can inhibit scavenger receptor uptake through the formation of peptide-protein nanoparticles. By inhibiting scavenger receptor uptake, DNA can be successfully hydrodynamically stimulated at times up to 12 hours post-delivery, demonstrating a longer circulatory half-life and suggesting a mechanism to explain how delayed hydrodynamic stimulation can achieve full level gene expression in the liver after the DNA has had time to circulate throughout the whole animal. Once a nonviral vector finds its target cell, it must still enter the cell through endocytosis and then escape the endosome before it becomes digested in the lysosome. Before the DNA cargo can be expressed, it must enter the nucleus. Nuclear entry in nondividing cells is a major barrier to efficient gene delivery. One method to over come this barrier is to avoid the need for nuclear entry altogether by delivering mRNA instead of DNA. mRNA can produce protein in the cytoplasm by finding a ribosome and initiating translation. However, it is even less stable in the bloodstream than DNA. We have produced an mRNA construct capable of high-level expression in the liver through hydrodynamic delivery. The PEGylated polyacridine peptides used to protect DNA were applied to mRNA and shown to enhance expression, allowing a 1μg dose of mRNA peptide polyplex to produce higher expression than an equal dose of DNA. The peptides were also shown to provide some protection against nuclease digestion in serum. This suggests that efficient, if transient, protein expression can be achieved through peptide protected mRNA delivery. However, DNA delivery is still desired for longer term expression, and the nuclear entry of DNA is still a problem. In an effort to help facilitate nuclear entry, the membrane disrupting enzyme phospholipase A2 was modified in several ways. The enzyme was conjugated with DNA binding peptides, nuclear localization peptides, and hepatocyte targeting oligosaccharides. Additionally, mutant forms of the enzyme were prepared in bacterial expression systems to achieve site-specific conjugation. Unfortunately, none of these efforts produced a useful tool for nuclear entry. The research presented in this thesis represents some progress toward the goal of nonviral gene delivery to the liver. Hopefully, some of this work will be useful in the development of new treatments and therapies to improve human health.