Formulation of Hyaluronidase Enzyme Sensitive Topical Nanomicrobicides for HIV Virus Transmission Prevention

Title from PDF of title page, viewed August 14, 2017Dissertation advisor: Bi-Botti C. YouanVitaIncludes bibliographical references (pages 159-187)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry, University of Missouri--Kansas City, 2015The objective of this dissertation is to design and optimize a nanoformulations (nanoparticle and nanofiber) delivery system loaded with anti-HIV topical microbicides for HIV prevention in women. In chapters 1 and 2, the overview of the problem, research objectives as well as the literature review of the technical and scientific background of this dissertation are introduced. In chapter 3, a study was designed to test the hypothesis that a triggered release of a topical anti-HIV microbicide (tenofovir: TFV) from hyaluronic acid based nanoparticles (HA NPs) can be achieved under the influence of hyaluronidase (HAase) enzyme. The Fractional Factorial Experimental Design (FFED) was employed to examine the formulation variables such as: molar concentrations of adipic acid dihydrazide (X₁) and 1-Ethyl-3-[3 dimethylaminopropyl]carbodiimide hydrochloride (X₂), volume of acetone (X₃) and reaction time (X₄), and their influence on the responses such as Y₁; particle mean diameter: PMD (nm), Y₂; polydispersity index: PDI and Y₃; zeta (ζ) potential: (mV). The cross-linking efficiency of NPs was characterized by Fourier Transform Infra-Red (FT-IR), and ¹³C-nuclear magnetic iv resonance (NMR) analyses. When formulated with X₁; 2.49 mM, X₂; 9.96 mM, X₃; 60 mL, X₄; 6 h, the HA-NPs exhibited a spherical shape with PMD, PDI, ζ potential, and drug loading of 70.6 ± 4.1 nm, 0.07 ± 0.02, -38.2 ± 2.8 mV, and 26.1 ± 1.2% w/w, respectively, (n = 3). Unlike for HA based gel, HAase notably triggered the drug release and HA degradation from the NPs after 24 h (~90% w/w and 65% w/w, respectively); whereas, in its absence, these values were ~39% w/w and 26% w/w, respectively. The NPs were non-cytotoxic to human vaginal VK2/E6E7 and End1/E6E7 cells and had no effect on Lactobacillus viability. These data suggested the possibility of using HA-NPs as a delivery system for intravaginal delivery of topical microbicides for the prevention of HIV transmission. In chapter 4, a study was designed to test the hypothesis that a stimuli-sensitive, safe and mucoadhesive thiolated hyaluronic acid (HA) based nanofibers (NFs) loaded with a topical vaginal microbicide (TFV) can be used for the prevention of HIV virus vaginal transmission in women. To test this, a novel thiolated sulfhydryl (-SH) group modified HA (HA-SH) was synthesized to fabricate the TFV loaded HA-SH-NFs (mean diameter ~75 nm) using electrospinning method. Sulfhydryl (-SH) group modified HA (HA-SH) were characterized for their size distribution, surface morphology, surface chemistry, crystallinity, mucoadhesion property, and in vitro drug release profile using size exclusion chromatography, powder X-ray diffraction, FT-IR, and ¹H-NMR analyses. Mucin interaction and ellipsometer measurements confirmed that mucoadhesion of HA-SH-NFs was increased compared to that of native HA polymer based on an increase in the size (~4 fold), thickness (~3 fold) and adsorbed mucin amount (~2 fold) after 3 h incubation of HA-SH-NFs with mucin. A triggered drug release (~87% w/w) from NFs (drug loading ~17% w/w) occurred after 1 h in the presence of seminal hyaluronidase enzyme. It was observed that in the absence of HAase, the drug release from NFs followed the Peppas kinetic model whereas, in the presence of HAase, NFs followed Weibull model. The HA-SH-NFs were non-cytotoxic to vaginal VK2/E6E7 and End1/E6E7 v cells and L. crispatus bacteria for 48 h. The results suggested that TFV loaded HA-SH-NFs templates developed in this study have the potential of vaginal delivery of topical microbicides for the prevention of HIV transmission. In chapter 5, in vivo evaluations of the developed HA-NPs and HA-SH-NFs were performed in female C57BL/6 mice. The histological analysis on the mice genital tract and other organs did not show any signs of damage upon once-daily administration of HA-NPs or HA-SH-NFs up to 7 days. Following 24 h exposure, HA-NPs or HA-SH-NFs did not show any significant immune (CD45) cell infiltration in mice vaginal tissues. The cytokines ((IL-1α, IL 1β, IL-6, IP-10, IL-7, MKC, TNF-α) levels (pg/mL) in cervicovaginal lavage and cervicovaginal tissues were not significantly changed compared to control mice data analyzed after 24 h. The cytokine results confirmed the non-immunogenicity of developed nanoformulations. The in vitro anti-HIV activity of HA-NPs and HA-SH-NFs was analyzed at the MOI of 10,000, 5,000, and 1,000 using a luciferase assay. The pseudotyped HIV virus particles were generated using lipofectamine plasmid transfection method. The size distribution, mean diameter (~128 nm), and titer (~3.07×10¹⁰) of pseudotyped virus particles was analyzed using nanoparticle tracking analysis measurements. The in vitro anti-HIV activity data showed that the TFV loaded HA-NPs and HA-SH-NFs were able to inhibit the pseudotyped HIV virus replication. Moreover, the results also confirmed that the structural integrity and anti-HIV activity of TFV was preserved after the nanofabrication processes. The in vivo results illustrated that these nanoformulations (HA-NPs and HA-SH-NFs) are promising delivery systems and offered a safe delivery of anti-HIV microbicide candidates. Overall, the data presented here highlight the applicability and potential of TFV loaded HA-NPs and HA-SH-NFs templates for the topical vaginal delivery of anti-HIV/AIDS microbicide candidates.Introduction -- Overview and pandemic of HIV infections -- Development of hyaluronidase sensitive tenofovir loaded hyaluronic acid based nanoparticles -- Fabrication of hyaluronidase sensitive tenofovir loaded hyaluronic acid based mucoadhesive nanofibers -- Preclinical safety and in vitro HIV efficacy evaluaitons of hyaluronidase sensitive tenofovir loaded hyaluronic acid based nanoformulations -- Summary, conclusion and future directions -- Appendi

Development and Preclinical Investigation of Physically Cross-Linked and pH-Sensitive Polymeric Gels as Potential Vaginal Contraceptives

This study explored the development of cross-linked gels to potentially provide a physical barrier to vaginal sperm transport for contraception. Two types of gels were formulated, a physically cross-linked iota-carrageenan (Ci) phenylboronic acid functionalized hydroxylpropylmethyacrylate copolymer (PBA)-based (Ci-PBA) gel, designed to block vaginal sperm transport. The second gel was pH-shifting cross-linked Ci-polyvinyl alcohol-boric acid (Ci-PVA-BA) gel, designed to modulate its properties in forming a viscoelastic, weakly cross-linked transient network (due to Ci gelling properties) on vaginal application (at acidic pH of ~3.5-4.5) to a more elastic, densely cross-linked (due to borate-diol cross-linking) gel network at basic pH of 7-8 of seminal fluid, thereby acting as a physical barrier to motile sperm. The gels were characterized for dynamic rheology, physicochemical properties, and impact on sperm functionality (motility, viability, penetration). The rheology data confirmed that the Ci-PBA gel was formed by ionic interactions whereas Ci-PVA-BA gel was chemically cross-linked and became more elastic at basic pH. Based on the screening data, lead gels were selected for in vitro sperm functionality testing. The in vitro results confirmed that the Ci-PBA and Ci-PVA-BA gels created a barrier at the sperm-gel interface, providing sperm blocking properties. For preclinical proof-of-concept, the Ci-PBA gels were applied vaginally and tested for contraceptive efficacy in rabbits, demonstrating only partial efficacy (40-60%). Overall, the in vitro and in vivo results support the development and further optimization of cross-linked gels using commercially available materials as vaginal contraceptives

The exciting potential of nanotherapy in brain-tumor targeted drug delivery approaches

Delivering therapeutics to the central nervous system (CNS) and brain-tumor has been a major challenge. The current standard treatment approaches for the brain-tumor comprise of surgical resection followed by immunotherapy, radiotherapy, and chemotherapy. However, the current treatments are limited in providing significant benefits to the patients and despite recent technological advancements; brain-tumor is still challenging to treat. Brain-tumor therapy is limited by the lack of effective and targeted strategies to deliver chemotherapeutic agents across the blood-brain barrier (BBB). The BBB is the main obstacle that must be overcome to allow compounds to reach their targets in the brain. Recent advances have boosted the nanotherapeutic approaches in providing an attractive strategy in improving the drug delivery across the BBB and into the CNS. Compared to conventional formulations, nanoformulations offer significant advantages in CNS drug delivery approaches. Considering the above facts, in this review, the physiological/anatomical features of the brain-tumor and the BBB are briefly discussed. The drug transport mechanisms at the BBB are outlined. The approaches to deliver chemotherapeutic drugs across the CNS into the brain-tumor using nanocarriers are summarized. In addition, the challenges that need to be addressed in nanotherapeutic approaches for their enhanced clinical application in brain-tumor therapy are discussed

Simultaneous quantitative determination of zidovudine and nevirapine in human plasma using isocratic, reverse phase high performance liquid chromatography

Purpose: To develop a sensitive and rapid reverse phase high performance liquid chromatography (HPLC) method for the measurement of the levels of zidovudine (ZVD) and nevirapine (NVP) in human plasma. Methods: Standard stock solutions for HPLC analysis were prepared by dissolving ZVD and NVP in methanol. In the HPLC measurement, sample detection was carried out at 246 nm using an ultraviolet (UV)-photo diode array (PDA) detector. Plasma sample pretreatment consisted of protein precipitation extraction with methanol. The compounds were separated using a mobile phase consisting of a pH 3.0 solution (obtained by adjusting the pH of water with orthophosphoric acid): acetonitrile (73:27 v/v) on a Phenomenex LUNA C18, column (250×4.6 mm i.d., 5µm) at a flow rate of 0.9 mL min. The total run time for the assay was 10.2 min-1 . The method was validated over the range of 300-9600 ng mL-1 and 200-6400 ng mL-1 for ZVD and NVP, respectively. Results: The lowest limits of quantification (LLOQ) and of detection (LOD) were 300 and 63 ng mL-1 for ZVD and 200 and 17 ng mLfor NVP, respectively. The method was found to be accurate, with accuracy ranging from -10.92 to +9.57 % and precise, with intra-day, inter-day as well as analyst to analyst precision of 0.68 to 9.38 %. Extraction recoveries of the drugs from plasma were 91.39, 95.01, 89.51 % for ZVD and 90.93, 93.26, 92.13 % for NVP, for LQC (low quality control), MQC (medium quality control) and HQC (high quality control) samples, respectively. Stability data revealed that the drugs were stable in plasma under various test conditions. Conclusion: This assay can be suitably used for the determination of zidovudine (ZVD) and nevirapine (NVP) in human plasma and should be useful in HIV clinical trials and clinical therapeutic drug monitoring (TDM) programs. It would also be potentially useful in the determination of pharmacokinetic profiles and in bioequivalence studies in HIV research

Cr modified Raman, optical band gap and magnetic properties of SnO2 nanoparticles

Pure and Cr (1, 3, 5, 7 and 10 at.%) doped SnO2 nanoparticles were synthesized in aqueous solution by a low cost chemical co-precipitation method without using any stabilizing agent. The effects of Cr doping on Raman, optical band gap and magnetic properties of SnO2 nanoparticles were investigated. Particle size is found to decrease with Cr doping into the SnO2 matrix which was confirmed by TEM. Besides of the fundamental mode of vibration, two additional peaks are also observed in Raman spectra which are correlated to Cr. The absorption spectra showed two peaks at 340 and 454 nm. The absorbance peak at 340 nm is assigned to the transition from valence band (VB) to conduction band (CB) and the peak at 454 nm was due to the transition from VB to mid gap energy level introduced by Cr. The optical band gap of undoped SnO2 nanoparticles is calculated to be 3 eV. With the doping of Cr in SnO2, band gap increases due to the decrease in particle size. The emission intensity is found to decrease with the increase in Cr doping due to the emission from CB to mid gap energy levels introduced by Cr between CB and VB. Undoped SnO2 nanoparticles show room temperature ferromagnetism due to the presence of defects and oxygen vacancies. The heavily doped SnO2 nanoparticles show paramagnetic nature due to the antiferromagnetic coupling between Cr and its nearest neighbour

Composite Nanoformulation Therapeutics for Long-Term Ocular Delivery of Macromolecules

The purpose of this investigation is to design and synthesize novel pentablock (PB) copolymer (PB-1: PCL–PLA–PEG–PLA–PCL) based nanoformulations suspended in a thermosensitive gelling copolymer (PB-2: mPEG–PCL–PLA–PCL–PEGm) termed as composite nanoformulation. The composite nanoformulation was prepared to provide a sustained delivery of macromolecules over a longer duration with negligible burst release effect. The delivery system was designed to be utilized for the treatment of posterior segment ocular diseases such as age-related (wet) macular degeneration, diabetic retinopathy, and diabetic macular edema. The novel PB copolymers were characterized for their functional groups by Fourier transform infrared spectroscopy, molecular weight and purity by ¹H NMR spectroscopy, and gel permeation chromatography. X-ray diffraction analysis was used to determine the crystallinity of copolymers. The size distribution of PB-1 nanoparticles (NPs) prepared using emulsification-solvent evaporation method was found to be ∼150 nm analyzed by nanoparticle tracking analysis. The % encapsulation efficiency and % drug loading were found to be 66.64% w/w ± 1.75 and 18.17% w/w ± 0.39, respectively, (<i>n</i> = 3). Different weight percentages (15 and 20 wt %) of the PB-2 copolymer have been utilized for <i>in vitro</i> release studies of IgG-Fab from composite nanoformulation. A negligible burst release with continuous near zero-order release has been observed from the composite nanoformulation analyzed up to 80 days. <i>In vitro</i> cell viability and biocompatibility studies performed on ocular (human corneal epithelial and retinal pigment epithelium) and mouse macrophage (RAW 264.7) cell lines showed that the synthesized PB copolymer based composite nanoformulations were safe for clinical applications. On the basis of the results observed, it is concluded that PB copolymer based composite nanoformulations can serve as a platform for ocular delivery of therapeutic proteins. In addition, the composite nanoformulation may provide minimal side effects associated with frequent intravitreal injections

Flow dynamics study of catalyst powder in catalytic cracking unit for troubleshooting

Gamma scanning and radiotracer applications are very effective and inexpensive tools to understand and optimize the process as well as troubleshoot the various types of problems in many chemical, petrochemical industries and refineries. These techniques are non-invasive; hence, the problems can be pinpointed online, which leads to reduce the downtime, schedule the shutdown and maintenance of the plant equipment, rendering huge economic benefits. In a leading refinery of India, the catalytic cracking unit (CCU) was malfunctioning. It was suspected by the refinery engineers that the catalyst powder was being carried over to the fractionator, which could have led to erosion of the fractionator column internals resulting in their rupture, and consequentially, to the fire hazard. To understand the flow behaviour of the catalyst powder and to ensure the mechanical integrity, catalyst accumulation and choking, both radiotracer study and gamma scanning of the CCU reactor was carried out. The reactor consists of a riser, three primary cyclones and three secondary cyclones. Gamma scanning of the reactor was carried out with the help of an automatic gamma scanner using 1.8 GBq of Co-60 sealed source. Results showed that the catalyst powder was accumulated in one of the secondary cyclones and uneven density distribution was observed in another secondary cyclone. The radiotracer study was carried out using the irradiated catalyst powder as a radiotracer, which contains 0.9 GBq of Na-24. The radiotracer was injected in the reactor through the specially fabricated injection system. Radiation measurement was done using the thermally insulated and collimated NaI(Tl) scintillation detectors located at various strategic locations coupled to a multi-detector data acquisition system. The data were mathematically analysed. It was confirmed that the catalyst powder was accumulated in one of the secondary cyclones with no flow downwards. This resulted in excess powder available to travel along with hydrocarbon towards fractionator. Since the quantity of powder released through the hydrocarbon outlet of CCU was higher than the designed value, the catalyst powder was observed in various zones of the fractionator. Mathematical modelling of the radiotracer data obtained at various locations corroborated the scanning results; also, the flow pattern was obtained. Partially blocked secondary cyclone showed plug flow with recirculation; normal working cyclone had plug flow behaviour and the vortex breaker showed parallel flow

Modelling tenofovir release kinetics from hyaluronidase-sensitive nanomedicine: A deterministic approach

Despite being convenient and practical, current nanomedicine (NM) release kinetic model remain unscalable, non-specific and less descriptive of the underlying physicochemical determinants. However, a deterministic mathematical modelling could overcome these limitations. In this study, we develop a model, based on a system of two differential equations (accounting for NM degradation and then drug release from degraded NM), which enable us to estimate per capita rate constant α (#NP degraded/hr) and β (Drug Amount Release /NP), the net effect of the nanomedicine (NE factor ɣ= α.β) and the controlled release index (φ, ratio of drug release to NP degradation). The model analysis conducted with tenofovir loaded hyaluronidase sensitive NM clearly shows the α factor significantly increased with triggering stimuli due to its enzymatic action on its substrate (hyaluronic acid). However, the β factor remained relatively unchanged, due to intrinsic physicochemical properties of the drug as limiting factor. The application of the ratio of NE factor analysis clearly enabled us to effectively screen among various nanoformulations and identified the best hyaluronidase-sensitive NM formulation, exhibiting the highest ratio (3.7-fold increase compared to no enzyme). The φ value confirmed the controlled release and stimuli sensitivity of the nanosystem. Moreover, the computed drug release rate (dM/dt) is consistent with other existing mathematical models (under valid assumption). The key advantages of this approach are i) relevancy to underlying physicochemical and biochemical process, ii) versatility and application to various NM kinetics, and iii) prediction of temporo-spatial distribution of the drug loaded nanocarrier (NC) that could potentially improve vitro/in vivo correlation study. This unique approach is applicable for a more specific and more meaningful/physicochemically relevant description of bioactive agent release from NM or NC for various applications