BIOMIMETIC STRATEGIES TO CONTROL THERAPEUTIC RELEASE FROM NOVEL DNA NANOPARTICLES

The inherent chemical, mechanical, and structural properties of nucleic acids make them ideal candidates for the formulation of tunable, personalized drug nanocarriers. However, none so far have exploited these properties for the controlled release of therapeutic drugs. In this dissertation, a biomimetic approach to controlling drug release is exhibited by specifically manipulating the architecture of novel, DNA nanoparticles to take advantage of drug binding mechanisms of action. Rationally designed DNA strands were immobilized on gold surfaces via a terminal thiol modification. Immobilized monomers can be manipulated to form distinct monolayer architectures including flat, folded, coiled, or stretched structures. Increasing the rate of folding is shown to restrict the diffusion of a surface-bound drug while upright architectures released drug at a 2 - 10 fold rate, depending on sequence length - using this strategy an over four-week release of dexamethasone was achieved. Furthermore, the release of an intercalating drug is controlled by exploiting sequence-specific affinities of the drug toward DNA. Here, using a high-affinity sequence and increasing the strand length a near zero-order release of daunomycin was achieved for up to 12 days. With this work, it is shown for the first time that the mechanisms of drug binding to nucleic acids can be utilized to produce highly controlled drug release from gold-core nucleic acid nanoparticles. These results will have a profound impact on the future design of novel, therapeutic nanocarriers

OIL/WATER NANOEMULSION BIODISTRIBUTION IN MICE UPON INTRAVENOUS ADMINISTRATION

The present study aimed to explore the biodistribution of O/W nanoemulsions (NE) upon intravenous administration. Three NEs were prepared with distinctive droplet sizes: SE (29 ± 1 nm), ME (214 ± 2 nm) and LE (883 ± 16 nm) without overlapping of the size distribution. Kolliphor® HS15 was used as the only surfactant for these three NEs, so that their droplets had similar surface structure. The NEs droplet size was stable under room temperature for minimum 3 days in phosphate buffer saline (PBS) and in mice plasma in vitro for 4-hour at 37°C. A lipophilic fluorescent dye, 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (DiI) was selected as the probe and loaded in the SE, ME and LE (designated thereafter as DSE, DME and DLE, respectively). A fluorometry for DiI was established with a linear range of 1.0-1000 ng/mL. The processing procedure and assay method for biological samples were developed. DiI extraction efficiency was 74.6-93.4%, depending on the tissues. For the biodistribution study, tumor-bearing mice received intravenous injection of DiI (2-5 mg/kg) in free solution (DS) or in the NEs via tail vein. The mice were sacrificed at sampling time points and the biological samples were assayed for DiI concentrations. DS manifested early tissues peak concentration (apparent Tmaxs at 0.5 h) followed by rapid decline, with tissue recovery mainly from the liver, spleen and lungs. DSE had a comparable plasma profile as DS but lower concentrations in the spleen and lungs as compared to the corresponding tissue profiles followed by the administration of DS. DME showed a sustained plasma circulation and a long-term non-specific higher tissue uptake with significant accumulation in the heart, lung, liver and spleen. DLE displayed a favorable accumulation in the RES organs including the lung, spleen, and liver. In conclusion, the present study demonstrates that O/W NE exhibits altered biodistribution upon intravenous administration. And these features may be utilized as a targeted drug delivery and drug redisposition strategy

PHASE-SEPARATING MICROBUBBLES FUNCTIONING AS VACCINE DEPOTS

Failure in receiving a booster for specific vaccines contributes to incomplete seroconversion, particularly in the developing world. Single injection vaccine technology could potentially be a solution such that health care personnel would not need to meet patients multiple times at designated points in time thereafter. The main challenge for single injection vaccine systems to date is controlling the stability of the antigen. to maintain the antigenic protein structure while in the physiological environment. We engineered a novel phase-separating microbubble technology which could function as an injectable depot which we hypothesize will enable us to control the microenvironment of the antigen for the durations required, in addition to controlling the antigen release time. We have successfully accomplished the following Main Specific Aims and subaims: Main Specific Aim 1: Synthesize polymers for microbubbles formation and Engineer methods for stabilizing Microbubbles: 1A: Synthesize PCL and PLGA library at different molecular weights and characterizing the polymers 1B: Synthesize acrylate polymers for microbubbles 1C: Engineer stable microbubble through UV cure and lyophilization 1D: Engineering the microbubbles to be stationary for maintaining sphere shape during the curing process and the inject of the cargo 1E: Engineering the cargo to be stationary within the polymeric microbubble to maximize the release time 1F: Quantify the diameter of the microbubble by varying syringe pump rate and comparing diameter pre- and post-lyophilization 1G: Quantify the angle of the micromotor for injecting cargo into the center of the microbubbles 1H: Engineer a self-contained lyophilization-capable system for the microbubbles Main Specific Aim 2: Engineering cargo release time of the microbubbles: 2A: Quantify how different molecular weights of PCL affect release time of the microbubbles 2C: Quantify how varying the microbubbles’ thickness of the shell controls the release time Main Specific Aim 3: Quantify stability of HIV and Hepatitis B antigens: 3A: Quantify how the HIV gp120/41 and HBsAg ayw antigens are stable in time in an aqueous environment versus in a cryo-protectant context at varying temperatures Our novel phase-separating technology which can form microbubble vaccine depots is a promising method to alleviate stability issues which hinders the single injection vaccine field. Enhancement of antigen stability in the microbubbles will be determined in future work

PHASE-SEPARATING MICROBUBBLES FUNCTIONING AS VACCINE DEPOTS

Failure in receiving a booster for specific vaccines contributes to incomplete seroconversion, particularly in the developing world. Single injection vaccine technology could potentially be a solution such that health care personnel would not need to meet patients multiple times at designated points in time thereafter. The main challenge for single injection vaccine systems to date is controlling the stability of the antigen. to maintain the antigenic protein structure while in the physiological environment. We engineered a novel phase-separating microbubble technology which could function as an injectable depot which we hypothesize will enable us to control the microenvironment of the antigen for the durations required, in addition to controlling the antigen release time. We have successfully accomplished the following Main Specific Aims and subaims: Main Specific Aim 1: Synthesize polymers for microbubbles formation and Engineer methods for stabilizing Microbubbles: 1A: Synthesize PCL and PLGA library at different molecular weights and characterizing the polymers 1B: Synthesize acrylate polymers for microbubbles 1C: Engineer stable microbubble through UV cure and lyophilization 1D: Engineering the microbubbles to be stationary for maintaining sphere shape during the curing process and the inject of the cargo 1E: Engineering the cargo to be stationary within the polymeric microbubble to maximize the release time 1F: Quantify the diameter of the microbubble by varying syringe pump rate and comparing diameter pre- and post-lyophilization 1G: Quantify the angle of the micromotor for injecting cargo into the center of the microbubbles 1H: Engineer a self-contained lyophilization-capable system for the microbubbles Main Specific Aim 2: Engineering cargo release time of the microbubbles: 2A: Quantify how different molecular weights of PCL affect release time of the microbubbles 2C: Quantify how varying the microbubbles’ thickness of the shell controls the release time Main Specific Aim 3: Quantify stability of HIV and Hepatitis B antigens: 3A: Quantify how the HIV gp120/41 and HBsAg ayw antigens are stable in time in an aqueous environment versus in a cryo-protectant context at varying temperatures Our novel phase-separating technology which can form microbubble vaccine depots is a promising method to alleviate stability issues which hinders the single injection vaccine field. Enhancement of antigen stability in the microbubbles will be determined in future work

Sustained release microparticles for pulmonary drug delivery.

In this study, several formulation approaches for generating sustained release (SR) microparticles suitable for pulmonary deposition are described. The model drug chosen for investigation was the hydrophilic β2-adrenoceptor agonist, Terbutaline Sulphate (TS), used in the treatment of asthma. A particular challenge to achieving suitable sustained release profiles arose from the high water solubility and ionised state of TS. Initial investigations focused on generating TS microcrystals, which would be subsequently coated with a SR excipient. A controlled crystallization method was developed in which TS was crystallized from an anti-solvent, which contained particle size restricting growth retardants. Significantly smaller crystals were obtained in the presence of growth retardants relative to crystallization without retardants. However, the smallest crystals obtained (3.6 μm) were too large for progression, as the application of a SR coat to such crystals would have increased particle size beyond that suitable for inhalation. The next investigation assessed TS release from a polysaccharide matrix particle containing molecularly dispersed active. Drug release was measured (HPLC) using a custom-built diffusion cell, designed to mimic release at the pulmonary epithelium. Release profiles showed that a degree of SR was possible from polysaccharide-based particles; although, SR was not sufficient for further development. Finally TS nanoparticles, obtained from an emulsion-template process, were encapsulated (spray-drying) within hydrophobic microparticles of respirable particle size. Several optimised formulations of this type provided promising in-vitro sustained release of the active at a variety of drug loadings and in a range of release media. The most useful SR excipient chosen for further development was hydrogenated palm oil, which was observed to coat the nanoparticles effectively. In-vitro deposition profiles were determined for a selection of formulations using an Andersen Cascade Impactor, and it was shown that deposition profiles were formulation-dependant and of size ranges suitable for pulmonary deposition

Physiologically based modelling of nanoparticle biodistribution and biokinetics

To predict the toxicity of nanoparticles (1-100 nm), it is crucial to understand their biokinetics i.e. how they are taken up, distributed, dissolved and removed from the body. Such information can be gained from biodistribution studies in animals. However, to make predictions for other types of nanoparticles, exposure conditions and species, including humans, extrapolations from such studies are required. Use of models, such as physiologically based pharmacokinetic (PBPK) models, makes extrapolations feasible, given that the models are sufficiently validated. In this thesis, a conceptual nanospecific PBPK model for intravenous administration to rats was developed and applied to different types of inert nanoparticles using experimental data from recent scientific publications (Papers I and II). The model represents systemic distribution and serves as a foundation for expansion to other species and other exposure routes (inhalation, dermal, oral). The PBPK simulations suggest that the model is able to describe the biokinetics of different types of inert nanoparticles given intravenously despite large differences in properties and exposure conditions. Our model is the first to include separate compartments for phagocytic cells and saturable phagocytosis. The simulations show that (1) phagocytosis needs to be incorporated in nano PBPK models, (2) the dose has a clear impact on biokinetics, but (3) further refinements are needed to better reflect processes such as agglomeration, corona formation and dissolution. The model was slightly modified to describe the biodistribution and biokinetics of nanoceria of different sizes and administered via other routes (Paper III). While the model could well predict the biokinetics after intravenous dosing, the predictions of inhalation, instillation and ingestion data were poor. The poor agreement may be partly due to low absorption via these routes, resulting in low nanoceria levels in tissues and organs, often close to or below the detection limit, in tissues. However, low absorption is hardly the only explanation, as the experimentally observed concentration time courses of nanoceria in tissues suggest that the biokinetics depend not only on the nanoparticle properties (size, coating) but also on the exposure conditions (dose, exposure route). The PBPK model was further developed to account for the complexity of inhalation exposure to nanoparticles (Paper IV). The modified model includes regional particle deposition in the respiratory tract, mucociliary clearance and phagocytosis in the lungs, olfactory uptake, and transport into the systemic circulation by alveolar wall translocation. The PBPK model described the biodistribution well and again suggested phagocytosis to be very important. The PBPK simulations were performed assuming that the nanoparticles are inert, i.e. do not dissolve or degrade in the body. However, when modelling the experimental data it seemed that the biokinetics might be better explained by introducing dissolution in the PBPK model. A related problem is that most experimental studies of metal nanoparticles use elemental analysis such as inductively coupled plasma mass spectrometry (ICP-MS). Such analyses do not discriminate between different forms of metal and therefore obscures the biokinetics. To test if gold nanoparticles dissolve in biological media, we developed an in vitro method to characterize dissolution of gold nanoparticles in contact with cell medium, macrophages and lipopolysaccharide (LPS)-triggered macrophages, simulating a disease state (Paper V). We demonstrated that gold nanoparticles are dissolved by cell medium and macrophages and even more so by LPS-triggered macrophages. The dissolution rate was higher for 5 nm than for 50 nm gold particles

Nanostructure empowers active tumor targeting in ligand-based molecular delivery

Altres ajuts: to EU COST Action CA 17140 and ICREA AcademiaCell-selective targeting is expected to enhance effectiveness and minimize side effects of cytotoxic agents. Functionalization of drugs or drug nanoconjugates with specific cell ligands allows receptor-mediated selective cell delivery. However, it is unclear whether the incorporation of an efficient ligand into a drug vehicle is sufficient to ensure proper biodistribution upon systemic administration, and also at which extent biophysical properties of the vehicle may contribute to the accumulation in target tissues during active targeting. To approach this issue, structural robustness of self-assembling, protein-only nanoparticles targeted to the tumoral marker CXCR4 is compromised by reducing the number of histidine residues (from six to five) in a histidine-based architectonic tag. Thus, the structure of the resulting nanoparticles, but not of building blocks, is weakened. Upon intravenous injection in animal models of human CXCR4 colorectal cancer, the administered material loses the ability to accumulate in tumor tissue, where it is only transiently found. It instead deposits in kidney and liver. Therefore, precise cell-targeted delivery requires not only the incorporation of a proper ligand that promotes receptor-mediated internalization, but also, unexpectedly, its maintenance of a stable multimeric nanostructure that ensures high ligand exposure and long residence time in tumor tissue

Proteins and peptides: non-invasive delivery

info:eu-repo/semantics/publishedVersio

Engineering secretory amyloids for remote and highly selective destruction of metastatic foci

Altres ajuts: to EU COST Action CA 17140Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format

A Liposomal Formulation Able to Incorporate a High Content of Paclitaxel and Exert Promising Anticancer Effect

A liposome formulation for paclitaxel was developed in this study. The liposomes, composed of naturally unsaturated and hydrogenated phosphatidylcholines, with significant phase transition temperature difference, were prepared and characterized. The liposomes exhibited a high content of paclitaxel, which was incorporated within the segregated microdomains coexisting on phospholipid bilayer of liposomes. As much as 15% paclitaxel to phospholipid molar ratio were attained without precipitates observed during preparation. In addition, the liposomes remained stable in liquid form at 4°C for at least 6 months. The special composition of liposomal membrane which could reduce paclitaxel aggregation could account for such a capacity and stability. The cytotoxicity of prepared paclitaxel liposomes on the colon cancer C-26 cell culture was comparable to Taxol. Acute toxicity test revealed that LD50 for intravenous bolus injection in mice exceeded by 40 mg/kg. In antitumor efficacy study, the prepared liposomal paclitaxel demonstrated the increase in the efficacy against human cancer in animal model. Taken together, the novel formulated liposomes can incorporate high content of paclitaxel, remaining stable for long-term storage. These animal data also demonstrate that the liposomal paclitaxel is promising for further clinical use