Browsing by Subject "Drug delivery"

Now showing 1 - 13 of 13
  • Thumbnail Image
    Item
    Amphiphilic polymers: crystallization-assisted Self-assembly and applications in pharmaceutical formulation
    (2013-04) Yin, Ligeng
    Amphiphilic polymers are macromolecules that simultaneously contain hydrophobic and hydrophilic components. These molecules not only attract much attention in academic research but also are important materials in industry. Application areas include detergency, oil field, paints, agriculture, food, cosmetics, and pharmaceutics. This dissertation highlights my efforts since the November of 2007 on three separate systems of amphiphilic polymers, which addresses both the fundamental self-assembly behavior in solution and applications in pharmaceutical formulation. Chapter 2 describes the self-assembled micelles in water that contain semicrystalline polyethylene (PE) as the core-forming material. Poly(N,N-dimethylacrylamide)-polyethylene (AE) diblock copolymers were chosen as the model system. An AE diblock copolymer with relatively low PE composition resulted in micelles with oblate ellipsoidal cores in water, in which crystalline PE existed as flat disks at the center and rubbery PE resided on both sides. In contrast, a control sample with a rubbery polyolefin as the hydrophobic component resulted in micelles with spherical cores in water. The morphology transition was ascribed to the crystallization of PE. The heat-assisted direct dissolution for sample preparation was identified as a stepwise "micellization-crystallization" procedure. In addition, the morphology of the aggregates exhibited much dependence on the composition of AE copolymers, and wormlike micelles and bilayered vesicles were obtained from samples with relatively high PE compositions. Chapter 3 demonstrates the precise synthesis of glucose-containing diblock terpolymers from a combination of anionic and reversible addition-fragmentation chain-transfer (RAFT) polymerizations. The resulting micelles exhibited excellent stability in several biologically-relevant media under in vitro conditions, including 100% fetal bovine serum. These particles may find applications as serum-stable nanocarriers of hydrophobic drugs for intravenous administration. Chapter 4 presents the development of novel cellulose derivatives as matrices in amorphous solid dispersions for improving the bioavailability of poorly water-soluble drugs in oral administration. Hydroxypropyl methylcellulose (HPMC) was modified with monosubstituted succinic anhydrides using facile anhydride chemistry, and the resulting materials simultaneously contained hydrophobic, hydrophilic, and pH-responsive moieties. Several HPMC esters of substituted succinates exhibited more effective crystallization inhibition of phenytoin under in vitro conditions than a commercial hydroxypropyl methylcellulose acetate succinate (HPMCAS). (341 words)
  • Thumbnail Image
    Item
    Antibody glycoengineering for drug delivery applications
    (2019-12) Sehgal, Drishti
    Monoclonal antibodies (mAbs) are frontline drugs for the treatment of many diseases including cancer 1 and rheumatoid arthritis 2 . In addition to their natural role as neutralizers of pathogens and toxins as well as in the recruitment of immune elements (complement, improving phagocytosis, antibody dependent cytotoxicity), they can be used as carriers for tumor-targeted delivery of therapeutic and diagnostic agents 3 . However, conjugation of drug or drug-encapsulated nanoparticles to antibodies can often result in reduced affinity of the antibody towards the target antigen. The overall objective of this thesis is to advance a new antibody glycoengineering technology that will allow for facile synthesis of antibody-based drug delivery systems. Most therapeutic mAbs are of the IgG class, which contains a glycosylation site in the Fc region at position 297 4 . In chapter 2, we investigated a glycoengineering strategy that enables the introduction of artificial azide groups at this glycosylation site without affecting their antigen affinity. This is based on the observation that glycosyltransferases present in mammalian cells can incorporate non-natural sugars (e.g., azido mannose) at glycosylation sites on an IgG molecule during the post translational modification. The azide groups in these artificial sugars are then available to react with alkynes through copper-catalyzed ‘click’ chemistry or with strained alkynes such as dibenzyl cyclooctyne (DBCO) allowing for biorthogonal, copper-free ‘click’ chemistry. Because the sugars are added reproducibly and at a site that does not affect antigen binding, the glycoengineering technology would overcome problems associated with traditional conjugation strategies. Using this approach, azide groups were introduced in anti-CD133 and anti-perlecan (AM6) antibodies. Further, the azide groups were available to react with various DBCO conjugates including fluorophores, drug molecules and nanoparticles. Importantly, the addition of artificial sugar and subsequent azide-alkyne reaction did not affect the affinity of the antibody for the target antigen. Antibody–drug conjugates (ADCs) have emerged as the next generation anticancer therapeutic agents. In chapter 3, glycoengineered AM6 antibody was used to generate an ADC with monomethyl auristatin E (MMAE) as the cytotoxic drug. The glycoengineering approach resulted in an ADC with a DAR of 2-3 drug molecules per antibody. The AM6- MMAE conjugate demonstrated enhanced cell kill in vitro and significantly improved anticancer efficacy in vivo compared to free MMAE. Similarly, in chapter 4, glycoengineered AM6 antibody was used to generate antibody conjugated polymeric nanoparticles loaded with paclitaxel. These perlecantargeted nanoparticles showed enhanced antitumor efficacy in vitro and in vivo in TNBC tumor models. Similarly, antibody conjugated nanoparticles showed enhanced antitumor efficacy in vitro and complete tumor growth inhibition in vivo in a non-muscle invasive bladder cancer model. We expect that this glycoengineering strategy will prove to be a unique platform technology that will have a significant impact on antibody-based therapeutics.
  • Thumbnail Image
    Item
    Chemically self-assembled antibody nanorings (CSANS): Design and characterization of an anti-CD3 IGM biomimetic.
    (2010-10) Li, Qing
    Based on our development of a highly efficient protocol for the chemically controlled self-assembling of protein nanorings, we have sought to exploit our methodology for engineering multivalent chemically self-assembled antibody-nanorings (CSANs) for tissue imaging and drug delivery. Two novel DHFR-DHFR-anti-CD3 scFv fusion proteins were constructed (13DDantiCD3 and 1DDantiCD3). In addition, the two DHFR cysteines were mutated to either alanine or serine to enhance correct folding. The protein was expressed in BL21 (DE3) cells, renatured with the SLS-based refolding protocol and purified by methotrexate affinity chromatography. Incubation of 13DDantiCD3 with the chemical dimerizer, bisMTX, resulted in almost exclusive formation of the bivalent CSANs, while incubation with 1DDantiCD3 resulted in formation of octavalent CSANs. Both antibody nanorings selectively blocked the killing of the CD3+ human T-leukemia HPB-MLT by a diphtheria-anti-CD3 immunotoxin. FACS analysis revealed nearly identical dissociation constants for both the selfassembled and parental monoclonal antibody and a 3-fold lower K d for the octavalent species. The chemically dimerized scFv's were shown to be stable in cell culture at 37°C and the dimerization was shown to be reversible by the addition of excess amounts of the non-toxic FDA approved DHFR antagonist trimethoprim. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (MAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells, and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e. 90%) of surface displayed T-cell receptor (TCR), only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15-25% less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental MAB. Comparative experiments with F(ab')2 derived from the MAB confirm that the activation of the T-cells by the MAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious affects of activation observed for MAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.
  • Thumbnail Image
    Item
    Development and characterization of aptamer-amphiphiles against fractalkine for targeted drug delivery
    (2013-12) Waybrant, Brett M.
    A foundation of modern diagnostics and therapeutics is the ability to non-covalently bind to a molecule of interest. These affinity molecules are behind a broad array of products ranging from therapeutics to HIV tests. Currently, antibodies are used as the affinity molecule. Despite the success of antibodies, alternatives are needed due to high development and production costs, and issues with stability. Aptamers are an exciting alternative to antibodies. Aptamers are short sequences of single stranded DNA or RNA that bind molecular targets with high affinity and specificity. Aptamers are inexpensive to produce, are very stable, have long shelf lives, and could potentially replace antibodies in a number of applications. One potential application of aptamers is targeted drug delivery. The goal of targeted drug delivery is to selectively deliver a therapeutic payload to the site of action thereby increasing efficacy and decreasing side effects. Fractalkine is a cell surface protein expressed at sites of inflammation. It is expressed on several types of cancerous tissues and it is involved in the patheogenisis of arthritis, asthma, and atherosclerosis. This work describes the development and characterization of an aptamer that binds fractalkine with high affinity. The aptamer was modified with a hydrophobic tail, creating an aptamer-amphiphile, for use in a model drug delivery vesicle called a liposome. The aptamer-amphiphile was optimized for a high affinity interaction with fractalkine by adding a spacer molecule between the aptamer headgroup and the hydrophobic tail. The optimized amphiphile had high affinity for fractalkine and self-assembled into micelles and an interesting nanotape morphology. Finally, as a proof of concept, the optimized aptamer-amphiphile was incorporated into a liposome and targeted to fractalkine expressing cells. This work highlights the development of aptamers as affinity ligands, and demonstrates their use as potential drug delivery agents.
  • Thumbnail Image
    Item
    Diblock copolymer stabilized nanoparticles for drug delivery via flash nanoprecipitation
    (2014-10) Han, Jing
    Cancer is one of the most challenge diseases to treat around the world. Drug delivery system, as one of the chemotherapeutic treatments has received enorrmous attention from researchers. This thesis is to develop amphiphilic diblock copolymer protected nanoparticles loaded with anti-cancer drug, with small size and high drug loading, to achieve selective drug delivery using EPR effect. Chapter 1 briefly describes the motivation and novelties of this research pursuit. Chapter 2 introduces a modified confined impingement jets mixer with dilution (CIJ-D mixer), using flash nanoprecipitation to produce nanoparticles made of hydrophobic drugs. The CIJ-D mixer was evaluated by the sizes of β-carotene nanoparticles at varied flow conditions compared to these made by multi-inlet vortex mixer. The CIJ-D mixer provides higher efficiency and easiness of handling for nanoparticle preparation. That is why CIJ-D mixer was used for all the work presented in the following chapters. In Chapter 3, we made the first attempt to produce PEG-b-PLGA protected paclitaxel loaded nanoparticles but failed, because paclitaxel is too hydrophilic to be captured in particles. Thus, a series of silicate ester derivatized paclitaxel were synthesized by Hoye research group and successfully encapsulated into nanoparticles. Several nanoparticle post-treatments, such as filtration, hollow fiber diafiltration, and ultracentrifugation were used and assessed, in order to purify nanoparticles. Lyophilization was found to induce nanoparticle aggregation due to the freezing process. The addition of sucrose as cryoprotectant was studied to prevent aggregation and recover nanoparticle. Chapter 4 focuses on developing in vitro drug release protocols, for more accurate quantification of highly hydrophobic paclitaxel prodrugs. Different dialysis devices were used such as dialysis tubes, dialysis cassettes, and dialysis mini capsules. Infinite sink and limited sink conditions were compared as well to provide sufficient concentration gradient across dialysis semi-permeable membrane. At last, a reverse drug release experimental protocol was customized to determine the remaining drug left in dialysis mini capsules while the sink condition was maintained by frequently refreshing buffer solution during in vitro drug release study. Chapter 5 mainly presents the pharmacokinetics of paclitaxel prodrug nanoparticles loaded with different silicate ester derivatives, at different pH, both inside nanoparticles and in buffer solution. Chapter 6 includes a series of Cryo-TEM images of nanoparticles collected at different time, such as fresh nanoparticles immediately after being prepared by CIJ-D mixer, nanoparticles after ultracentrifugation, after lyophilization, 0hr, and 24 hr during drug release study. These images not only showed a reverse liner relation of average particle size and hydrophobicity of the loaded drug, but also displayed a core-shell internal structure of nanoparticles prepared via flash nanoprecipitation and potential particle disassembly after 24hr drug release. Finally, Chapter 7 summarizes the key results and conclusions obtained from previous chapters, lessons learned from mistakes and failures, and future directions for this project, in order to prepare nanoparticles with better controlled size and drug release kinetics and to understand deeply on nanoparticle formation and release mechanisms.
  • Thumbnail Image
    Item
    Formulation and delivery of polymeric nanoparticle-assisted vaccine against melanoma
    (2015-04) Niu, Lin
    Poly (lactide-co-glycolide) (PLGA) nanoparticle (NP) is a widely used biodegradable carrier for drug and vaccine delivery. This thesis focused on the formulation, delivery and efficacy of PLGA NP for its potential application in melanoma immunotherapy. To enable reliable PLGA NP formulation for clinical use such as vaccination, lyophilization is the method of choice to manufacture dry NP dosage form. A major risk of the lyophilization product development for NPs is the irreversible NP aggregation due to freezing and drying stress. Based on real-time imaging, freezing stress could be attributed to freeze-concentration of NPs. Cryo-scanning electron microscopy (cryo-SEM) revealed individual NP separately embedded in the freeze-concentrate interstitial space of the sucrose formulation, leading to corroborative support for the "particle isolation" hypothesis of cryo-protection. Various sphere packing models were investigated to guide the rational design of cryo-/lyo-protectant containing NP formulations. To facilitate precise intradermal delivery of NP formulation for vaccination, microneedle array-mediated administration was utilized to deliver large volume of NPs into the skin. The majority of the infused PLGA NPs were retained locally. A PLGA NP vaccine formulation delivered intradermally elicited robust humoral and cellular immunity. Antigen-loaded NP formulation triggered quicker and stronger high affinity antibody responses compared to the soluble antigen formulation. Vemurafenib, a selective inhibitor of BRAF V600E, induces apoptotic melanoma cell death and remarkable tumor burden reduction. However, drug resistance invariably occurs. Novel TLR7/8 agonists were encapsulated in PLGA particulate formulation as immunostimulatory nanoparticles (ISNP) to boost immune response against drug-resistant melanoma. NP-mediated intracellular delivery contributed to enhanced dendritic cell activation in vitro and antigen-specific CD8+ T cell proliferation in vivo. The prophylactic vaccination using NP-assisted whole tumor cell formulation prolonged the survival of mice challenged with melanoma. To take advantage of the clearance of melanoma antigens by immune system in the context of BRAF inhibition, an ISNP-assisted in situ whole tumor cell vaccination strategy was investigated using BRAF V600E positive mouse SM1 melanoma cells. Despite the suppressed tumor growth, no survival benefit was observed in this therapeutic vaccination model.
  • Thumbnail Image
    Item
    Improving delivery of elacridar to enhance efficacy of molecularly-targeted agents in treatment of glioblastoma
    (2012-12) Sane, Ramola Vishwas
    Treatment of glioblastoma with new molecularly-targeted agents has been largely ineffective in clinical trials. Many of these molecularly-targeted agents are substrates for the efflux transporters P-gp and BCRP, and therefore, one of the reasons for the lack of efficacy could be the limitations to drug delivery to the target site. P-gp and BCRP are efflux transporters that are expressed at the blood-brain barrier, which acts as a protective mechanism and prevents chemotherapeutics from reaching the brain parenchyma. P-gp and BCRP are also expressed at the tumor cell surface. These two sequential barriers could restrict the access of chemotherapeutics to the target site and therefore could reduce their efficacy. The main objective of this work was to overcome these sequential barriers by use of a pharmacological inhibitor of P-gp and BCRP, elacridar. The ultimate aim was to develop a chronic dosage regimen of a molecularly-targeted agent with elacridar as an adjuvant to enhance drug delivery to the brain in preclinical models of glioma. We demonstrated that the bioavailability of elacridar is limited due to its poor physicochemical properties. We also showed that the distribution of elacridar into the brain is limited by the presence of P-gp and BCRP and is governed by a saturable efflux process that can be overcome by increasing the dose of elacridar. We developed a microemulsion formulation of elacridar that improved its bioavailability several-fold and allowed us to decrease the dose of elacridar required to show an inhibitory effect. We examined the effect of elacridar as an adjuvant to erlotinib in treatment of tumor-bearing mice. It was found that while the co-administration of elacridar definitely improved the brain distribution of erlotinib, it did not offer any advantage in improving overall survival of the tumor bearing animals. These observations show that improving the distribution of a single molecularly targeted agent may not be sufficient in order to effectively target a heterogenous tumor such as glioma. To effectively treat an aggressive disease such as glioblastoma, a combination of drugs that target a number of growth pathways; combined with a pharmacological inhibitor of transporters could help formulate an effective strategy to target tumor cells.
  • Thumbnail Image
    Item
    Improving The Delivery Of Novel Molecularly-Targeted Therapies For The Treatment Of Primary And Metastatic Brain Tumors
    (2019-01) Gampa, Gautham
    Tumors in the brain are challenging to diagnose and are associated with poor survival outcomes. Brain tumors are difficult to treat, in part, due to restricted drug delivery across the blood-brain barrier (BBB). Although the BBB is compromised in some regions of brain tumors, the degree of disruption is not uniform and certain tumor locations have a functionally intact BBB. A critical component of BBB that restricts entry of therapeutics into brain is active efflux. The objective of this work is to examine brain distribution of novel molecularly-targeted therapies, including evaluation of influence of P-gp and Bcrp-mediated efflux at the BBB, assessment of spatial heterogeneity in drug distribution to brain tumors, and comparison of unbound (active) drug exposures with in vitro efficacy. Ispinesib is a KIF11 inhibitor that inhibits both tumor proliferation and invasion in glioblastoma (GBM). We demonstrate that ispinesib has a limited brain delivery due to efflux by P-gp and Bcrp, and ispinesib delivery is heterogeneous to areas within a tumor in a GBM model. Furthermore, predicted unbound-concentrations in brain were less than in vitro cytotoxic concentrations, suggesting that delivery may limit in vivo efficacy. Also, pharmacological inhibition of efflux transport (elacridar co-administration) improves brain delivery of ispinesib, and future studies will evaluate if enhanced delivery will improve efficacy. CCT196969, LY3009120 and MLN2480 are panRAF inhibitors with minimal paradoxical activation of MAPK pathway and may overcome resistance observed with BRAF inhibitor therapy in melanoma. MEK inhibition is used in combination with BRAF inhibitors to delay resistance. E6201 is a potent MEK inhibitor with a unique macrocyclic structure. While brain distribution of panRAF inhibitors is limited by efflux, E6201 has a favorable brain distribution profile and interacts minimally with P-gp and Bcrp. The delivery of E6201 is variable to regions of tumor in an intracranial melanoma model. However, predicted unbound-concentrations in brain achieve levels higher than in vitro cytotoxic concentrations for LY3009120 and E6201, suggesting possible efficacy in melanoma brain metastases. Future studies evaluating in vivo efficacy in preclinical models will reveal the utility of selected compounds in brain tumor treatment, and if improved delivery translates to superior efficacy.
  • Thumbnail Image
    Item
    Multi-segmented magnetic nanowires as multifunctional theranostic tools in nanomedicine
    (2015-07) Sharma, Anirudh
    Nanomedicine is the development and use of nanostructured materials that have unique diagnostic and therapeutic effects owing to their size and structure. Current research efforts in this field have focused on eliminating cancer using novel nanoparticle-based therapeutics, e.g. heat induced tumor destruction via light or AC magnetic field excitation. This Ph.D. research presents a novel multifunctional nanoparticle, the multilayered magnetic nanowire, as a unique, robust and effective diagnostic and therapeutic (theranostic) platform for applications in translational nanomedicine. Here, multilayered magnetic nanowires are synthesized de novo using high-throughput electrochemical deposition in nanoporous templates. The presented applications in nanomedicine exploit the fiber-like shape of the nanowire, which is used advantageously for magnetic multiplexing, drug-delivery and synthesis of artificial biomaterials through self-assembly. This research has addressed important engineering questions pertaining to the design and synthesis of the nanowire, including shape, size, composition, magnetic properties, surface functionalization, nanoparticle aggregation and integration of this technology with various biomedical applications. Further, the ensuing cellular and immunological responses were examined in-depth using a variety of techniques including cell-based assays and microscopy in order to address biocompatibility, immunogenicity, inflammatory properties, cytotoxicity and proliferative effects. The proven success of the multilayered nanowire in these applications makes it an indispensable diagnostic and therapeutic tool in nanomedicine and regenerative technology.
  • Thumbnail Image
    Item
    Prenylated Chemically Self-Assembled Nanorings: A Versatile Platform for Macro-Chemical Biology
    (2022-02) Wang, Yiao
    Macro-chemical biology uses chemical methods and biomacromolecules to study and manipulate biological systems at the cellular level, and biomacromolecules capable of manipulating cell fates have been demonstrated exceedingly valuable for assorted fundamental research and therapeutic applications. Protein conjugates, as a type of chemically engineered hybrid macromolecules, are synthesized through conjugations of proteins with functional molecules of diverse types and have been exploited to manipulate cell functions in various aspects, including regulation of intercellular interactions, intervention in intracellular biological pathways, and termination of cell proliferation. Although effective in numerous pre-clinical studies, the clinical translation of therapeutic protein conjugates remains challenging and hindered by some limitations, which in turn underlines some ideal features for designing clinically desirable protein conjugates. Hence, to meet these challenges, our group has constructed a versatile macromolecular platform with self-assembling protein-lipid conjugates, namely, prenylated chemically self-assembled nanorings (CSANs), for macro-chemical biology studies. This multivalent system has exhibited exceptional stability, function reversibility, target cell selectivity, and broad utility, and was shown to effectively regulate cell fates for multiple biomedical applications. We first used the prenylated CSANs as a universal non-genetic system to stably modify cell surface and demonstrated they can mediate reversible cell-cell interactions for fundamental research and adoptive cell therapy. Thereafter, we discovered the CSANs can specifically transfer from the CSAN-modified cell to the target cell during cell-cell interactions, and such intercellular CSAN transfer is dependent on specific ligand-receptor engagement. Therefore, with fluorescent dyes conjugated to the farnesylated CSANs through click reactions, the CSAN assisted cell-cell cargo transfer (C4T) was utilized as a tool to record cell-cell interactions. Furthermore, by conjugating functional oligonucleotides and cancer drugs to the farnesylated CSANs, we were able to manipulate cell functions by the C4T approach. Finally, the cancer drug MMAE was conjugated to the CSANs to form a targeted drugdelivery system for the treatment of EGFR-positive cancer. The MMAE-loaded anti-EGFR CSANs manifested notable cytotoxicity and selectivity against EGFR-positive cancer cells and were also found to provoke immunogenic death of the cancer cells. Altogether, these promising results demonstrate that the prenylated CSANs are a versatile platform applicable to a variety of fundamental research and therapeutic purposes.
  • Thumbnail Image
    Item
    Trafficking and efficacy of cationic polymers as DNA vaccine carriers and anti-cancer agents
    (2013-06) Panus, David
    The potential of DNA vaccines for treatment of diseases such as HIV and cancer are overwhelming, due to the fact that DNA vaccines can activate both a cell-mediated (T-cell) and humoral (antibody) immune response. However, the most commonly occurring problem of DNA vaccines is limited transgene delivery and expression. Currently, much effort has focused on designing an optimal polymer system that is stable, can protect and deliver DNA, as well as offer high transgene expression. Unfortunately, the ability of polymer based systems to produce robust gene expression, have yet to show substantial improvement. The major obstacle hindering successful transgene expression can be attributed to the interactions of the polymer-DNA complex with the subcellular environment. Therefore, we focused on understanding the structure-functional relationship of a well-defined simple polymer based system and how they might lead to improved transgene expression. First, we investigated the effect of polymer molecular weight and backbone structure on transgene expression as it pertains to subcellular trafficking. Second, we focused on the relationship between polymer-DNA complexes and different dendritic cell-types as a function of maturation state. Lastly, we looked into how further modification of a cationic polymer can lead to elevated cytotoxicity and use as an anti-cancer agent. The results from this work can be used as a design template to improve the overall subcellular trafficking and efficacy of cationic polymer based DNA vaccines or anti-cancer agents.
  • Thumbnail Image
    Item
    A Universal Delivery Platform : Near Infra-Red Activated Nanoparticles for Drug, Peptide, and Small Molecule Delivery
    (2018-10) Shin, Jeongeun
    A generic delivery platform that could deliver any biomolecule, independent of its chemical constitution, would be a significant advance not only for treatments of cancers and genetic diseases, but also for researches in cell biology and neuroscience. At present, introducing endogenous proteins, genetic materials, or other non-native species into cancer or other cells has been tested via mechanical force-driven methods (electroporation, microinjection, and membrane deformation) and nanoparticle-mediated methods (lipid, polymeric, or inorganic nanoparticles). However, mechanical force-driven delivery methods cause loss of cell viability since they brutally breach cell membrane to form transient pores and require high concentration of molecules to be delivered, compared to nanoparticle-mediated methods. Contents release from current nanoparticle-mediated delivery platforms is hard to control, because release kinetics is determined by the physicochemical properties of nanoparticles. Therefore, it is hard to trigger contents at a desired time and location. In addition, delivering functional proteins or genetic materials to cells requires bypassing the cell membrane, which is done most efficiently via endocytosis. However, endosome escape is still the most difficult to overcome bottleneck for drug, protein or nucleic acid delivery by nanoparticles. Understanding the ways in which cancer cells respond to different local concentrations of small molecules and probing the signaling pathways thus initiated often requires sub-micron and sub-millisecond resolution of physiological processes. The challenge is to perturb the cell environment with a concentration jump of a physiological ligand, cofactor or antagonist, with timing and spatial dimensions that mimic the physiological process. At present, such fast intervention is the realm of “caged” ATP, calcium and other small molecules. However, small molecule delivery via caged compounds requires each bioactive requires the chemical synthesis of its own “cage”. The use of high intensity ultraviolet (UV) light causes damage to surrounding cells and has a relatively shallow level of effectiveness in the human body. To address these issues by delivering functional proteins or small biologically active molecules to individual cells in culture, and rapidly manipulating the chemical or biological environment within and in the vicinity of a particular cell, we proposed a new platform technology based on the interactions of plasmon-resonant hollow gold nanoparticles (HGN) with physiologically friendly, highly penetrating near infrared (NIR) light. NIR light is easy to use, manipulate and target with subcellular resolution using either a conventional two-photon microscope or our customized laser set up, which opens new ways to induce events and control the level of impact in specific cell populations, even subcellular sites. HGN are thin gold shells with a hollow, water-filled core, showing the unique and highly tunable optical properties, so-called Localized Surface Plasmon Resonance (LSPR). The LSPR allows the HGN-absorbance maximum can be easily tuned to 600 – 900 nm, NIR light, by adjusting the ratio of the shell thickness to the nanoparticle diameter. The advantage of NIR is that it can penetrate several centimeters of soft tissues without showing any significance harmful effect on tissues. In previous works, the HGN synthesis, which involves the galvanic exchange of gold on a silver nanoparticle template, provides a relatively polydisperse population, which broadens the absorption maximum. We have developed new methods of creating monodispersed hollow gold nanoparticles in different sizes (25 – 40 nm) and shapes (nanospheres and nanocubes). Moreover, we optimized the reaction to synthesize highly monodispersed as small as 10 nm, with temperature control. The irradiation of picosecond NIR light pulses onto HGN shows the unique transient heat transfer dynamics. The pulse duration is significantly faster that the rate of heat dissipation into the surrounding fluid (nanosecond to microsecond). This confines the energy to the HGN, leading to a transient increase in HGN temperature above metltng point of gold-silver alloy (~ 1000 ℃) depending on the light intensity. At these temperatures, gold-thiol bonds that are used to conjugate thiol-labeled protein or siRNA to the HGN surface, are broken, releasing small molecule, protein, or siRNA cargo from the HGN. In the following nanoseconds, the hot HGN nucleates vapor nanobubbles, which rapidly grow and collapse, similar to cavitation bubbles, and induce mechanical disruption of endosome, cell or liposome membranes. Nanobubbles provide efficient endosome escape with cell-resolution selectivity, and then dissipate, leaving no toxic materials behind. Direct cargo release to the cytoplasm makes for high efficiency; reducing biomolecule concentration required more than an order of magnitude compared to commercially available chemical reagents for intracellular delivery. We detected the generation of bubble around the HGN as the evidence of fast (picoseconds) NIR absorption. We then compared the threshold energy for bubble generation in different sizes, shapes, LSPR wavelength and surface coverage of nanoparticles. The minimum fluence for nanobubble generation decreased with HGN size at a fixed LSPR wavelength, unlike solid gold nanoparticles that require an increased fluence with decreasing size. We also show that he laser fluence of NIR pulses increases as the irradiation wavelength moves off the HGN LSPR. As HGN concentration decreases, the threshold fluence necessary to induce transient vapor nanobubbles increases due to light localization through multiple scattering. However, surface treatment (citrate stabilized or thiol-linked polyethylene glycols ranging from 750 to 5000 molecular weight) made no significant difference on the threshold fluence. We then examined contents release via HGN delivery platform. We have developed HGN conjugated with thiol-labeled liposomes. As nanocarriers, liposomes can encapsulate almost any water-soluble biologically active molecule by confining high concentration. A major benefit of this technique is the universal mechanism of liposome contents release via nanobubble rupture following pulsed NIR light triggering: any molecule will be released by liposome rupture, so release rates, timing, laser fluence, etc. will be similar for all compounds of interest. By modifying the laser fluence, HGN properties, and liposome membrane composition, we can alter the energy threshold for triggering release, enabling delivery of multiple agents at different times and locations, which is impossible with current liposome or caged compound technologies. Chemically disparate calcium, ATP, and carboxyfluorescein (CF) are all released at near 100 % efficiency from liposomes within msec. For a given HGN tethered to the liposome, the threshold energy is lowest at the wavelength corresponding to the maximum adsorption wavelength of the HGN; the threshold energy increases as the wavelength of the NIR light moves away from the maximum. This allows us to create liposomes that can release at different laser fluences so that we could control release rates and windows of each biomolecule in a mixture independently, by delivering two species or even changing the order of release. In this way, we can release one compound at one place and time, then a second compound at the same place at a different time simply by modulating the laser energy. This independent release has never been demonstrated before. This independent, sequential and timely release of multiple contents shows a potential of our technology for an application in combination therapies that require multiple injections to fight against multiple drug resistance (MDR), which is still a bottle neck for cancer treatments. We presented three different applications to show the versatility of our technology. We remotely triggered calcium release from liposomes in an alginate hydrogel and induced spatially patterned alginate gelation. We also succeed to entrap mammalian cells at desired sites in alginate gel via NIR light triggered calcium release. We then showed intracellular drug delivery using our HGN tethered liposomes. Cisplatin, a prostate and ovarian cancer drug, was introduced into PC3 prostate cancer cells and showed enhanced cell killing depending on HGN size and laser fluence, compared to free cisplatin. We also demonstrated that enhanced therapeutic efficacy of cisplatin was observed when cisplatin containing liposomes were irradiated in a flow, compared to cisplatin release that happens on static cell culture condition. At last, we showed toxic KLAK peptide and Cre recombinase release from the HGN surface via nanobubble generation. With our system, we decreased IC50 of KLAK peptide by a million-fold and increased cell killing as increasing laser fluence. Upon NIR pulse irradiation, delivery of Cre recombinase into HeLa cells via nanobubble generation edited genes in cells and modified cells to express red fluorescence as an indicator of successful cell modification. Delivery of cisplatin, KLAK peptide, and Cre recombinase via our HGN platform was performed in our customized microfluidic system that shows a potential of our technology for high-throughput gene-editing, cell modification and immunotherapy for treatments of cancer and genetic diseases. This innovative delivery platform would be powerful medical and scientific tools to advance modern targeted biomolecule delivery technologies and resolve challenges for loading, delivery, and release of multiple drugs.
  • Thumbnail Image
    Item
    Volume transitions in gels with biomedical applications: Mechanics and electrodiffusion.
    (2010-07) Micek, Catherine Ann
    In this thesis, mathematical models for gels are developed and analyzed using both analytical and numerical approaches. The work is motivated by two biomedical applications: body implantable devices such as artificial bone implants and a drug delivery device designed by Siegel et al. [14, 41, 42, 62]. The mathematical structure of the models depends on the device being studied: the former application is an equilibrium problem focusing on mechanical effects, whereas the latter is a dynamical problem focusing on chemomechanical effects. Both types of models are considered in this work. The mechanical equilibrium model presented is suitable for gel problems in both the mixing or separating regime. For mixing regime problems, the existence of minimizers for a convex energy is established. The Euler-Lagrange equilibrium equations for this model are equations of nonlinear elasticity, and the Stokes elasticity mixed finite element method is developed for the linearized Euler-Lagrange equations. The Stokes formulation is used to numerically simulate the effects of confinement and temperature changes with the software FEniCs for an artificial bone implant. For the separating regime model, the existence of minimizers for a non-convex energy is established following the proof first presented in [61]. The dynamical model presented is an electrochemical model derived from balance laws for mechanics and chemistry. The primary goal in the analysis of this model is to model a cyclic gel volume phase transition using chemomechanical coupling. Two issues are addressed: the origin of the volume phase transition and modeling a mechanically realistic cycling mechanism. Following the studies of Horkay et al. in physiology [34, 35], the volume phase transition is formulated as higher order terms from the Flory-Huggins mixing energy. After a careful examination of the chemical and mechanical governing equations, the cycling mechanism is modeled as a non-monotone mechanical stress for which hysteresis is inherently present. The mechanical emphasis of the model is an alternative approach to the chemical emphasis found in the models of Siegel et al.