Drug Delivery Devices
Yilong Fu, Lai-Chun Ong, Sudhir H. Ranganath, Lin Zheng, Irene Kee, Wenbo Zhan, Sidney Yu, Pierce K. H. Chow, Chi-Hwa Wang. A dual tracer 18F-FCH/18F-FDG PET imaging of an orthotopic brain tumor xenograft model. PLOS ONE. 2016,2,4;11(2): e0148123
Abstract
Early diagnosis of low grade glioma has been a challenge to clinicians. Positron Emission Tomography (PET) using 18F-FDG as a radio-tracer has limited utility in this area because of the high background in normal brain tissue. Other radiotracers such as 18F-Fluorocholine (18F-FCH) could provide better contrast between tumor and normal brain tissue but with high incidence of false positives. In this study, the potential application of a dual tracer 18F-FCH/18F-FDG-PET is investigated in order to improve the sensitivity of PET imaging for low grade glioma diagnose is based on a mouse orthotopic xenograft model. BALB/c nude mice with and without orthotopic glioma xenografts from U87 MG-luc2 glioma cell line are used for the study. The animals are subjected to 18F-FCH and 18F-FDG PET imaging, and images acquired from two separate scans are superimposed for analysis. The 18F-FCH counts are subtracted from the merged images to identify the tumor. Micro-CT, bioluminescence imaging (BLI), histology and measurement of the tumor diameter are also conducted for comparison. Results show that there is a significant contrast in 18F-FCH uptake between tumor and normal brain tissue (2.65±0.98), but with a high false positive rate of 28.6%. The difficulty of identifying the tumor by 18F-FDG only is also proved in this study. All the tumors can be detected based on the dual tracer technique of 18F-FCH/18F-FDG-PET imaging in this study, while the false-positive caused by 18F-FCH can be eliminated. Dual tracer 18F-FCH/18F-FDG PET imaging has the potential to improve the visualization of low grade glioma. 18F-FCH delineates tumor areas and the tumor can be identified by subtracting the 18F-FCH counts. The sensitivity was over 95%. Further studies are required to evaluate the possibility of applying this technique in clinical trials.
P Davoodi, M.P. Srinivasan, C.H. Wang. Synthesis of Intracellular Reduction-Sensitive Amphiphilic polyethyleneimine and poly(e-caprolactone) Graft Copolymer for On-demand Release of Doxorubicin and p53 plasmid DNA. Acta Biomaterialia. 39,79-93(2016) .
Abstract
This study aims to present a new intelligent po lymeric nano-system used for combining chemotherapy with non-viral gene therapy against human cancers. An amphiphilic copolymer synthesized through the conjugation of low molecular weight polyethyleneimine (LMw-PEI) and poly(ε-caprolactone) (PCL) via a bio-cleavable disulfide linkage was successfully employed for the simultaneous delivery of drug and gene molecules into target cells. Compared to the conventional PCL copolymerization pathway, this paper represents a straightforward and efficient reaction pathway including the activation of PCL-diol hydroxyl end groups, cystamine attachment and LMw-PEI conjugation which are successfully performed at mild conditions as confirmed by FTIR and 1H NMR. Thermal, morphological characteristics as well as biocompatibility of the copolymer were investigated. The copolymer showed great tendency to form positively charged nanoparticles (∼163.1 nm, +35.3 mV) with hydrophobic core and hydrophilic shell compartments implicating its potential for encapsulation of anti-cancer drug and plasmid DNA, respectively. The gel retardation assay confirmed that the nanoparticles could successfully inhibit the migration of pDNA at ∼5 nanoparticle/pDNA w/w. The in vitro cytotoxicity tests and LDH assay revealed that the cationic amphiphilic copolymer was essentially non-toxic in different carcinoma cell lines in contrast to branched PEI 25K. Moreover, the presence of redox sensitive disulfide linkages provided smart nanoparticles with on-demand release behavior in response to reducing agents such as cytoplasmic glutathione (GSH). Importantly, confocal microscopy images revealed that in contrast to free Dox, the nanoparticles were capable of faster internalizing into the cells and accumulating in the perinuclear region or even in the nucleus. Finally, the co-delivery of Dox and p53-pDNA using the copolymer displayed greater cytotoxic effect compared with the Dox-loaded nanoparticle counterpart as revealed by cell viability and Caspase 3 expression assay. These results suggest the copolymer as a promising candidate for the development of smart delivery systems.
P Davoodi, W.C. Ng, W.C. Yan, M.P. Srinivasan, C.H. Wang. Double-walled Microparticles-embedded Self-crosslinked, Injectable, and Anti-bacterial Hydrogel for Controlled and Sustained Release of Chemotherapeutic Agents. ACS Applied Materials & Interfaces 8(35), 22785-22800 (2017)
Abstract
First-line cancer chemotherapy has been prescribed for patients suffered from cancers for many years. However, conventional chemotherapy provides a high parenteral dosage of anticancer drugs over a short period, which may cause serious toxicities and detrimental side effects in healthy tissues. This study aims to develop a new drug delivery system (DDS) composed of double-walled microparticles and an injectable hydrogel for localized dual-agent drug delivery to tumors. The uniform double-walled microparticles loaded with cisplatin (Cis-DDP) and paclitaxel (PTX) were fabricated via coaxial electrohydrodynamic atomization (CEHDA) technique and subsequently were embedded into injectable alginate-branched polyethylenimine. The findings show the uniqueness of CEHDA technique for simply swapping the place of drugs to achieve a parallel or a sequential release profile. This study also presents the simulation of CEHDA technique using computational fluid dynamics (CFD) that will help in the optimization of CEHDA’s operating conditions prior to large-scale production of microparticles. The new synthetic hydrogel provides an additional diffusion barrier against Cis-DDP and confines premature release of drugs. In addition, the hydrogel can provide a versatile tool for retaining particles in the tumor resected cavity during the injection after debulking surgery and preventing surgical site infection due to its inherent antibacterial properties. Three-dimensional MDA-MB-231 (breast cancer) spheroid studies demonstrate a superior efficacy and a greater reduction in spheroid growth for drugs released from the proposed composite formulation over a prolonged period, as compared with free drug treatment. Overall, the new core–shell microparticles embedded into injectable hydrogel can serve as a flexible controlled release platform for modulating the release profiles of anticancer drugs and subsequently providing a superior anticancer response.
WC Yan, XJ Ong, KT Pun, YD Tan, VK Sharma, YW Tong, CH Wang. Preparation of tPA-loaded Microbubbles as Potential Theranostic Agents: A Novel One-Step Method via Coaxial Eletrohydrodynamic Atomization Technique. Chemical Engineering Journal. 307, 168-180 (2017).
Abstract
In the present work, we demonstrated for the first time a simple method for the fabrication of drug-loaded Microbubbles (MBs) by a single step via coaxial eletrohydrodynamic atomization (CEHDA). As a proof of concept, a therapeutic agent (tissue plasminogen activator, tPA) and two types of shell materials (phospholipid and bovine serum albumin, BSA) were selected to produce tPA-entrapped MBs. Investigation using fluorescein isothiocyanate (FITC) labelled tPA revealed that the tPA-loaded MBs were successfully fabricated in a one-step procedure and the tPA was located in the shell layer for both the BSA and lipid MBs. By optimization of the operating conditions in terms of voltage, core / shell flow rate ratio as well as tPA volume ratio, minimum bubble sizes for tPA-BSA and tPA-lipid MBs were obtained. The fabricated tPA-BSA MBs was ∼41 μm in mean diameter while ∼41% of the tPA-lipid MBs ranged from 3-6 μm and ∼36% of them ranged from 6-9 μm under optimal operating conditions. Sensitivity analysis on the effects of key process parameters was also performed to guide design and manipulation of bubble sizes. The investigation of gas phase showed that the usage of sulphur hexafluoride (SF6) as the core can enhance the stability of tPA-lipid bubbles. The presented one-step method displayed great flexibility for producing tPA-loaded MBs and thus can potentially serve as a new tool to generate/engineer tPA-bubbles for applications in ischemic stroke therapy.
Q. Xu, H. Qin, Z. Yin, J. Hua, D. W. Pack, C.H. Wang, “Coaxial electrohydrodynamic atomization process for production of polymeric composite microspheres”, Chem. Eng. Sci. 104, 330-346 (2013).
Abstract
Polymeric composite microspheres consisting of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(d,l-lactic acid) (PDLLA) shell layer were successfully fabricated by coaxial electrohydrodynamic atomization (CEHDA) process. Process conditions, including nozzle voltage and polymer solution flow rates, as well as solution parameters, such as polymer concentrations, were investigated to ensure the formation of composite microspheres with a doxorubicin-loaded PLGA core surrounded by a relatively drug-free PDLLA shell layer. Various microsphere formulations were fabricated and characterized in terms of their drug distribution, encapsulation efficiency and in vitro release. Numerical simulation of CEHDA process was performed based on a computational fluid dynamics (CFD) model in Fluent by employing the process conditions and fluid properties used in the experiments. The simulation results were compared with the experimental work to illustrate the capability of the CFD model to predict the production of consistent compound droplets, and hence, the expected core–shell structured microspheres.
CEHDA process for producing uniform composite core–shell structured microspheres
Transmitted light, scanning electron and confocal micrographs depicting doxorubicin-loaded core–shell structured microspheres. The green color shows the distribution of doxorubicin. Scale bar=50 µm
Representative compound droplets that are produced during stable cone–jet mode at different time points under various nozzle voltages. The time interval is 0.5 ms. The red, green and blue colors represent the core, shell and air phases, respectively. In all cases, the core/shell flow rates (1.0/3.5 ml/h) and the nozzle-to-collector distance (15 cm) are maintained. Scale bar=100 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Y. Cui, Q. Xu, P K.H. Chow,D. Wang, C.H. Wang, “Transferrin-conjugated magnetic silica PLGA nanoparticles loaded with doxorubicin and paclitaxel for brain glioma treatment”, Biomaterial, 34, 8511-8520 (2013).
Abstract
The effective treatment of malignant brain glioma is hindered by the poor transport across the blood–brain barrier (BBB) and the low penetration across the blood-tumor barrier (BTB). In this study, transferrin-conjugated magnetic silica PLGA nanoparticles (MNP-MSN-PLGA-Tf NPs) were formulated to overcome these barriers. These NPs were loaded with doxorubicin (DOX) and paclitaxel (PTX), and their anti-proliferative effect was evaluated in vitro and in vivo. The in vitro cytotoxicity of drug-loaded NPs was evaluated in U-87 cells. The delivery and the subsequent cellular uptake of drug-loaded NPs could be enhanced by the presence of magnetic field and the usage of Tf as targeting ligand, respectively. In particular, cells treated with DOX-PTX-NPs-Tf with magnetic field showed the highest cytotoxicity as compared to those treated with DOX-PTX-NPs-Tf, DOX-PTX-NPs, DOX-PTX-NPs-Tf with free Tf. Thein vivo therapeutic efficacy of drug-loaded NPs was evaluated in intracranial U-87 MG-luc2 xenograft of BALB/c nude mice. In particular, the DOX-PTX-NPs-Tf treatment exhibited the strongest anti-glioma activity as compared to the PTX-NPs-Tf, DOX-NPs-Tf or DOX-PTX-NPs treatment. Mice did not show acute toxicity after administrating with blank MNP-MSN-PLGA-Tf NPs. Overall, MNP-MSN-PLGA-Tf NPs are promising carriers for the delivery of dual drugs for effective treatment of brain glioma.
(A) and (B) TEM images of MNP-MSN-PLGA-Tf NPs; (C) XPS spectra of MNP-MSN-PLGA NPs and MNP-MSN-PLGA-Tf NPs.
(A) Fluorescence images showing biodistribution of NPs in tumor-bearing mice after 2 h tail vein injection of MNP-MSN-PLGA-Tf NPs (control), CM-NPs and CM-NPs-Tf. The blue circle indicates the location of the brain tumor; (B) Variation of mice body weight as a function of post-treatment time; (C) Histological examination of tumor slices harvested from mice after 20 days post-treatment. The blue circle represents the necrotic regions in the tumor interior. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
C. Lei, Y. Cui , L. Zheng, P.K.H. Chow, C.H. Wang, “Development of a gene/drug dual delivery system for brain tumor therapy: Potent inhibition via RNA interference and synergistic effects”, Biomaterial, 34(30), 7483-7494 (2013)
Abstract
Malignant brain tumors are characterized by three major physiological processes: proliferation, angiogenesis, and invasion. Traditional cytotoxic chemotherapies (e.g. Paclitaxel) control the tumor by blocking growth and proliferation mechanisms, but leave angiogenesis and invasion unchecked. We identified Matrix metalloproteinase-2 (MMP-2), an essential proteinase regulating brain tumor invasion and angiogenesis, as one of the therapeutic target. A designer RNAi plasmid was developed, and complexed with the gene carrier polyethylenimine (PEI), in an effort to specifically suppress MMP-2 expression in tumor cells. The gene and a cytotoxic drug Paclitaxel were then dual-encapsulated in PLGA based submicron implants to achieve a sustained release of both agents. Potent inhibition effects on MMP-2 mRNA and protein expression, in vitro cell angiogenesis and invasion were demonstrated both on the PEI/DNA nanoparticles alone, and on the PEI/DNA nanoparticles embedded in microfibers. Most importantly, through in vivo test on intracranial xenograft tumor model in BALB/c nude mice, it was proved that the gene/drug dual delivery microfibers are able to impose significant tumor regression compared with single drug delivery microfibers and commercial drug treatment, showing evidence for synergistic therapeutic efficacy.
SEM images of microfiber sheets immersed in PBS for pore formation study for 3 different dosage forms. A. H10; B. H20; C. M10. Top panel: before immersion; Middle panel: 7 days; Bottom panel: 14 days.
Bioluminescence imaging results to reveal tumor therapeutic efficacy in vivo. (A). Representative BLI images of intracranial U87MG-luc2 xenograft in mice from sham, placebo, drug implant and drug/gene dual implant groups over time. Luciferase expressing tumor cells are displayer in the brain with a red-blue color bar, red color indicating the highest bioluminescence intensity. (B). Intracranial U87MG-luc2 tumor progression profile during treatment, measured as normalized bioluminescence intensity for various groups. n = 3–5 animals.
F.J. Wang, T.K.Y. Lee and C.H. Wang , “PEG Modulated Release of Etanidazole from Implantable PLGA/PDLA Discs”, Biomaterials, 23 3555-3566 (2002).
Abstract
With the development of technology, radiosensitizers have been worked out for the treatment of cancer since they could greatly reduce the radiation dose, resulting in less pain and side effects to the patients. But many radiosensitizers have short half-life, degrade easily in vivo, and hence a large oral dose is needed. On the other hand, drug dose must be controlled within a range where toxicity could be avoided. In this way, the efficacy is always restrained by the amount of drug used and the ability to enter the target site. Therefore it is necessary to develop an efficient system to deliver the needed radiosensitizer to the cancer site. As a member of nitroimidazole radiosensitizer, etanidazole can deplete intracellular glutathione and inhibit glutathione transferases, thereby enhancing sensitivity to the radiation and alkylating agents. Its preliminary results have shown great promise for the cancer treatment. In our research, this drug is being used to observe the release characteristics such as radiation response and control the release rate. We are also interested in developing an intermittent system and a multi-component drug system, and simulation of the drug distribution in the tumor.
Release profile of etanidazole from small PLGA65: 35 discs using different PEG types and loadings. Disc diameter = 5 mm, thickness = 1 mm, compression pressure= 2 ton/m^2, retention time = 3 minutes.
Scanning electronic microscope pictures of PLGA65: 35 microspheres incorporated with different loadings of PEG8000 a) 5% PEG b) 10% PEG.
T. H. Lee, J. Wang and C.H. Wang, “Double-walled microspheres for the sustained release of a highly water soluble drug: characterization and irradiation studies”, J. Controlled Release, 83, 437-452 (2002).
Abstract
Composite double-walled microspheres with biodegradable poly(-lactic acid) (PLLA) shells and poly(-lactic–co-glycolic acid) (PLGA) cores were fabricated with highly water-soluble etanidazole entrapped within the core as solid crystals. This paper discusses the characterization, in vitro release and the effects of irradiation on this class of microsphere. Through the variation of polymer mass ratios, predictable shell and core dimensions could be fabricated and used to regulate the release rates. A direct and simple method was devised to determine the composition of the shell and core polymer based on the different solubilities of the polymer pair in ethyl acetate. A distribution theory based on solubility parameter explains why highly hydrophilic etanidazole has the tendency to be distributed consistently to the more hydrophilic polymer. Release profiles for normal double-walled samples have about 80% of drug released over 10 days after the initial time lag, while for irradiated double-walled samples, the sustained release lasted for more than 3 weeks. Although sustained release was short of the desired 6–8 weeks required for therapy, a low initial burst of less than 5% and time lags that can be manipulated, allows for administration of these microspheres together with traditional ones to generate pulsatile or new type of releases. The effects of irradiation were also investigated to determine the suitability of these double-walled microspheres as delivery devices to be used in conjunction with radiotherapy. Typical therapeutic dosage of 50 Gy was found to be too mild to have noticeable effects on the polymer and its release profiles, while, sterilization dosages of 25 kGy, lowered the glass transition temperatures and crystalline melting point, indirectly indicating a decrease in molecular weight. This accelerated degradation of the polymer, hence releasing the drug.
SEM photographs of cross-sectional view of double-walled microspheres. (A) Etanidazole loaded microsphere, PLLA–PLGA (2:1); (B) unloaded microsphere, PLLA–PLGA (1:1); (C) unloaded microsphere, PLLA–PLGA (2:1); (D) unloaded microsphere, PLLA–PLGA (2.5:1).
Cumulative release profile of etanidazole from double-walled microspheres. Mass ratios (2:1) (▲) ; 1:1 (♦). (B) Plot of cumulated release Qt versus t1/2 to show diffusion controlled drug release. (■) PLLA–PLGA (2:1, w/w); (▲) PLLA–PLGA (1:1, w/w).
M. Zhang, Z. Yang, L.L. Chow, C.H. Wang, “Simulation of drug release from biodegradable polymeric microspheres with bulk and surface erosions”, J. Pharm. Sci., 92, 2040-2056 (2003).
Abstract
New models are developed to account for the kinetics of drug release from porous, biodegradable polymeric microspheres under the schemes of bulk erosion and surface erosion of the polymer matrix, respectively. Three mechanisms of drug release, namely, drug diffusion, drug dissolution, and polymer erosion jointly govern the overall release process. For bulk erosion, the model incorporates an erosion term into the dissolution and diffusion equation and is solved numerically for various boundary conditions. Dissolution and erosion are defined in the model by introducing three equations which take into account the drug concentration in the liquid phase, virtual solid phase, and effective solid phase. For surface erosion, drug concentrations in liquid and solid phases are defined and a substitution is introduced to convert the moving-boundary problem to a fixed-boundary problem. The resulting differential equations are solved simultaneously to obtain the concentration profile in the liquid and solid phases, respectively. Numerical solutions are provided to illustrate the effects of drug dissolution constant, drug diffusion coefficient, and erosion rate constant. In general, increasing erosion rate, diffusivity, dissolution, and decreasing particle radius enhance the drug release rate. Predictions from the models are also compared with experimental data to verify their validity and possible improvements are proposed.
J. Wang, K. M. Chua and C. H. Wang, “Stabilization and encapsulation of human immunoglobulin G into biodegradable microspheres”, J. Colloid & Interfacial Sci., 271, 92-101 (2004).
Abstract
The instability of protein during preparation, storage, and release has become a major concern in recent years in the encapsulation of proteins into biodegradable polymers for controlled release systems. The present investigation was performed to study the mechanism of degradation of human immunoglobulin G (IgG) in double emulsion and solid-in-oil-in-water (S/O/W) encapsulation processes. The stabilizing effects of various excipients during the period of protein atomization using spray freeze-drying and subsequent encapsulation into polylactide-co-glycolide (PLGA) microspheres were explored. The size-exclusion high-performance liquid chromatography (SEC-HPLC) results showed that ultrasonication did not change the primary structure of IgG significantly. However, enzyme-linked immunosorbent assay (ELISA) revealed that the subsequent double-emulsion solvent evaporation process denatured nearly 80% of the total amount of IgG. This was possibly due to the adsorption, unfolding, and aggregation of IgG at the water/organic solvent interface. Both mannitol and trehalose could stabilize IgG during spray freeze-drying, with over 90% retention of its molecular integrity and immunoactivity, which were verified using SEC-HPLC and ELISA. Solid protein microparticles were further entrapped into monolithic-type microspheres of PLGA using the S/O/W method. FTIR results suggested that the incomplete release that is often observed in the formulation of controlled protein release systems may be due to the degradation or aggregation of protein in the solid polymer matrix.
SEM observation of IgG particles made by spray freeze-drying with different formulations. (a) Pure IgG, (b) zinc acetate:IgG = 1:1, (c) 5 times dilution with mass ratio of Zn:IgG = 1:1, (d) 10 times dilution with mass ratio of Zn:IgG = 1:13.7.
Scanning electron microscopy pictures of PLGA 50:50 microspheres of different preparation methods. (a) Double emulsion, (b) S/O/W
L. Ding, T. Lee and C. H. Wang, “Fabrication of monodispersed Taxol-loaded particles using electrohydrodynamic atomization”, J. Controlled Release, 102, 395-413 (2005).
Abstract
In the fabrication of controlled drug release matrix, monodispersed particle sizes are usually preferred as they give a more uniform and precisely controlled release profile. Electrohydrodynamic atomization (EHDA) [J.C. Ijsebaert, K.B. Geerse, J.C.M. Marijnissen, J.W.J Lammers, P. Zannen, Electro-Hydrodynamic atomization of drug solutions for inhalation purposes, J. Appl. Physiol. 91(2001), 2735-2741.] is a method that can potentially produce particles with very low polydispersity. In the present work, Taxol-loaded poly-caprolactone (PCL) particles were fabricated using EHDA. Effort was undertaken to investigate the cause of the low yield of EHDA and to improve it to around 80%. This was achieved by increasing the ventilation and properly discharging the residual charges on the particulate cake at the filter paper. A phase Doppler particle analyzer (PDPA) was used to detect the spray modes of EHDA and the optimum operating conditions were determined. With differential scanning calorimetry (DSC), the uniformity of drug and polymer matrix is investigated. Moderate zeta potential values together with laser confocal micrographs give a possible explanation for the in vitro cell uptake data. These results are substantiated by a reasonably high encapsulation efficiency (EE) and sustained release profiles over 1-month period. The EHDA method is shown to be a potentially suitable technique to prepare close to monodispersed drug release particles.
SEM images. (a) overall view of PCL particles (collected from filter); (b) overall view of PCL particles fabricated from spray drying technique; (c) overall view of PCL particles (collected from side wall and then went through ultrasonic stirring and freeze drying treatment); (d) overall view of PCL particles after 45 days in vitro release; (e) morphology of PCL particle collected from filter; (f) morphology of PCL particle collected from side wall; (g) morphology of PCL particle collected from filter. PCL/DCM concentration: 7.5% (g/ml) for particles in panels a–f. (g) Same as (f) except PCL/DCM concentration is 3% (g/ml). Drug loadings for all samples were 1%, except sample b; other samples were fabricated using the EHDA method.
R. Lin, L. S. Ng and C. H. Wang, “In vitro study of anticancer drug doxorubicin in PLGA-based microparticles”, Biomaterials, 26, 4476-4485 (2005).
Abstract
Doxorubicin (DOX), also known as adriamycin, is an anthracycline drug commonly used in cancer chemotherapy. Unfortunately, its therapeutic potential has been restricted by its dose limited cardiotoxicity and the resistance developed by the tumor cells to the molecule after some time of treatment. One way to overcome these problems is to encapsulate the drug in poly (d, l-lactide-co-glycolide) (PLGA) microparticles. This paper investigates the release characteristics of DOX from polymeric carriers fabricated using the spray-drying technique. The encapsulation efficiency, size and morphology of the various polymeric devices were also determined. In order to improve the release characteristics, Pluronic P105 (PLU) and poly (l-Lactide) (PLLA) are individually used in combination with PLGA. Finally, a cytotoxicity test was performed using Glioma C6 cancer cells to investigate the cytotoxicity of DOX delivered from PLGA microparticles. It has been found that the cytotoxicity of DOX to Glioma C6 cancer cells is enhanced when DOX is delivered from PLGA polymeric carrier.
(i) 
(ii)
(i) SEM pictures showing the morphology of composite microparticles fabricated by varying PLLA/PLGA ratio and drug loading from 1% to 3%. (A) PLLA/PLGA=20/80, 2% drug loading (C2); (B) 30/70, 2% (C3); (C) 50/50, 2% (C4); (D) 30/70, 3% (C6). (ii) Confocal fluorescence microscopy showed the time dependence of Glioma C6 cells uptake of spray-dried microparticles: (A) 30 min, (B) 60 min, (C) 120 min, (D) cells incubated with free dye for 60 min.
P. K. Naraharisetti, M. D. N. Lew, Y.C. Fu, D. J. Lee and C. H. Wang, “Gentamicin-loaded discs and microspheres and their modifications: characterization and in vitro release”, J.Controlled Release, 102, 345-359 (2005).
Abstract
Osteomyelitis is an infection of the bone, and successful treatment involves local administration for about 6 weeks. Gentamicin is a very hydrophilic drug and tends to come out into the water phase when microspheres are fabricated using solvent evaporation method. Hence, spray drying is an option, and it was observed that the release rate tends to be fast when the particle size is small and large particles cannot be prepared by spray drying. In an effort to get better encapsulation efficiency and release rate, we have worked on the possibility of compressing the microspheres into discs and modifying the porosity of the discs by using biocompatible materials like polyethylene glycol (PEG) and calcium phosphates and also on the fabrication of double-walled and composite microspheres. In the case of microspheres, two methods of fabrication both based on solvent evaporation method were employed. The two polymers used are poly-L-lactide (PLLA) and copolymers of poly-DL-lactic-co-glycolic acid (PLGA). One method is based on the spreading coefficient theory for the formation of double-walled microspheres by using single solvent, while the other is based on the property of PLLA not being soluble in ethyl acetate (EA). Characterization to check if the microspheres formed are double-walled was performed. The fabrication method where two solvents, dichloromethane (DCM) and ethyl acetate, were used gave double-walled microspheres, while the other where only dichloromethane was used gave composites. The double-walled microspheres were smaller in size compared to the composites, which were in the range of 100–600 μm. This can be attributed to the difference in the fabrication procedure. We were able to achieve better encapsulation efficiencies of more than 50% and slower release rates, which lasted for about 15 days. It was observed that size played a major role in the encapsulation efficiency and release rates. The possibility of achieving better results by studying the effect of concentration of polymer in solvent and the effect of using different polymers was investigated.
In vitro release discs made by blending beta-tricalcium phosphate (at 60%) and microspheres. TCP is beta-tricalcium phosphate. (b) In vitro release discs made by blending hydroxylapatite(at 60%) and microspheres. HAP is hydroxylapatite. (c) In vitro release from beta-tricalcium phosphate and microspheres blended discs at 40% and 30% calcium phosphate with PLGA 50:50 microspheres. T—beta-tricalcium phosphate.
J. Xie and C. H. Wang, “Self-Assembled Biodegradable Nanoparticles Developed by Direct Dialysis for the Delivery of Paclitaxel”, Pharmaceutical Research, 22(12), 2005.
Abstract
The main objective of this study was to obtain self-assembled biodegradable nanoparticles by a direct dialysis method for the delivery of anticancer drug. The in vitro cellular particle uptake and cytotoxicity to C6 glioma cell line were investigated.
Self-assembled anticancer drugsVpaclitaxel-loaded poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLA) nanoparticlesVwere achieved by direct dialysis. The physical and chemical properties of nanoparticles were characterized by various state-of-the-art techniques. The encapsulation efficiency and in vitro release profile were measured by high-performance liquid chromatography. Particle cellular uptake was studied using confocal microscopy, microplate reader, and flow cytometry. In addition, the cytotoxicity of this drug delivery system was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on C6 glioma cell line to predict the possible dose response of paclitaxel-loaded PLGA and PLA nanoparticles.
PLGA and PLA nanoparticles with or without vitamin E tocopherol polyethylene glycol succinate (TPGS) as an additive were obtained, in which the sustained release of paclitaxel of more than 20 days was achieved. The coumarin6-loaded PLGA and PLA nanoparticles could penetrate the C6 glioma cell membrane and be internalized. The cytotoxicity of paclitaxel-loaded nanoparticles seemed to be higher than that of commercial Taxol\ after 3 days incubation when paclitaxel concentrations were 10 and 20 µg/ml.
Direct dialysis could be employed to achieve paclitaxel-loaded PLGA and PLA nanoparticles, which could be internalized by C6 glioma cells and enhance the cytotoxicity of paclitaxel because of its penetration to the cytoplasm and sustained release property.
Confocal fluorescence images of C6 glioma celss with coumarin6-labeled nanoparticles (200-300nm) with different exposure times. (a,b) PLGA nanoparticles incubated for 1h; (c,d) PLGA nanoparticles incubated for 2h; (e,f) PLA nanoparticles incubated for 2h. The images were collected from the fluorescein isothiocyanate channel and neutral red channel simultaneously.
J. Xie, J. C. M. Marijnissen, C. H. Wa,ng “Microparticles developed by electrohydrodynamic atomization for the local delivery of anticancer drug to treat C6 glioma in vitro”, Biomaterials (2006) 27: 3321-3332.
Abstract
This study aims to fabricate biodegradable polymeric particles by electrohydrodynamic atomization (EHDA) for applications in sustained delivery of anticancer drug – paclitaxel to treat C6 glioma in vitro. Controllable morphologies such as spheres, donut shape and corrugated shape with sizes from several tens of microns to hundred nanometers of particles were observed by scanning electron microscopy (SEM) and field emission electron microscope (FSEM). The differential scanning chromatography (DSC) study indicated that paclitaxel could be either in an amorphous or disordered-crystalline phase of a molecular dispersion or a solid solution state in the polymer matrix after fabrication. The x-ray photon spectroscopy (XPS) result suggested that some amount of paclitaxel could exist on the surface layer of the microparticles. The encapsulation efficiency was around 80% and more than 30 days in vitro sustained release profile could be achieved. Cell cycling results suggested that paclitaxel after encapsulation by EHDA could keep its biological function and inhibit C6 glioma cells in G2/M phase. The cytotoxicity of paclitaxel-loaded biodegradable microparticles to C6 glioma cells could be higher than Taxol® in the long-term in vitro tests evaluated by MTS assay. The drug delivery devices developed by EHDA in this study could be promising for the local drug delivery to treat malignant glioma.
SEM images of typical samples of EHDA microparticles
Xie, C. H. Wang, Electrospun micro- and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro. (2006) 23(8):1817-1826.
Abstract
Purpose. The present study aims to develop electrospun PLGA-based micro- and nanofibers as implants for the sustained delivery of anticancer drug to treat C6 glioma in vitro.
Methods. PLGA and an anticancer drug – paclitaxel-loaded PLGA micro- and nanofibers were fabricated by electrospinning and the key processing parameters were investigated. The physical and chemical properties of the micro- and nanofibers were characterized by various state-of-the-art techniques, such as scanning electron microscope and field emission scanning electron microscope for morphology, x-ray photoelectron spectroscopy for surface chemistry, gel permeation chromatogram for molecular weight measurements and differential scanning calorimeter for drug physical status. The encapsulation efficiency and in vitro release profile were measured by high performance liquid chromatography. In addition, the cytotoxicity of paclitaxel-loaded PLGA nanofibers was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide MTT) assay on C6 glioma cell lines.
Results. PLGA fibers with diameters of around several tens nanometers to 10µm were successfully obtained by electrospinning. Ultrafine fibers of around 30nm were achieved after addition of organic salts to dilute polymer solution. The encapsulation efficiency for paclitaxel-loaded PLGA micro- and nanofibers was more than 90%. DSC results suggest that the drug was in the amorphous state in the polymeric micro- and nanofibers. In vitro release profiles suggest that paclitaxel sustained release was achieved for more than 60 days. Cytotoxicity test results suggest that IC50 of paclitaxel-loaded PLGA nanofibers was around 600µg/well (for 24 wells plate).
Conclusions. Electrospun paclitaxel-loaded biodegradable micro- and nanofibers may be promising for the treatment of brain tumour as alternative drug delivery devices.
Xie, C. H. Wang, Microencapsulation of living cells using electrospray in dripping mode. Journal of Colloids and Interface Science, (2007) In press.
Abstract
Entrapment of living cells in microcapsules is to protect the encapsulated cells from the host’s immune system, which can be used as drug delivery vehicles, immunotherapies and engineered tissues. The main objective of the present study was to investigate the droplet formation and to better develop mono-dispersed microencapsulation of living cells with controllable size. The uniformity of microencapsulation size was realized by performing electrospray in the dripping mode and also stabilized by an additional ring electrode. Reduction of droplet diameter and increase in the dripping frequency were observed with increasing applied voltage to the nozzle using a conventional electrospray setup. The vibration of the needle was found to reduce when high voltage was applied to the nozzle. With increasing voltage applied to the ring electrode, the dripping frequency was found to decrease with the formation of slightly larger sizes of droplets. Hep G2 cell line was taken as the model cell line for encapsulation in calcium alginate microbeads. Relatively uniform microbeads could be achieved when operating under low flow rates with high voltages applied to the nozzle by using a conventional electrospray setup. In contrast, uniform microbeads can not be obtained using a similar setup under high flow rates unless the ring electrode is applied with a voltage to stabilize the electrospray in the dripping mode. In this modified electrospray, microbeads with narrow size distribution and slightly larger size can be obtained even for cases under high flow rates. Phase contrast microscope images showed that the diameter of microcapsules from around 200 µm to 2 mm could be finely tuned by adjusting various operating parameters.
Fabrication of controlled release devices using supercritical fluid techniques Lee Lai Yeng
Abstract
Many routes exist for fabrication of micro-/ nanoparticles using supercritical fluids. These include rapid expansion of supercritical solutions (RESS) and supercritical antisolvent (SAS). The most common supercritical fluid used in these processes is Carbon dioxide (CO2). CO2 is a great choice for processing pharmaceutical products due to its relatively low supercritical temperature (Tc = 31.1 oC and Pc = 73.8 bar) and it is also non toxic. In the immediate vicinity of the critical point, the density of the supercritical CO2 is around 0.4g/ml. For reduced pressures > 2, the density of SC CO2 is comparable to that for liquid CO2. It is this liquid-like density that enables many materials to be dissolved in several orders of magnitude greater than that predicted by ideal gas considerations.
In particular, we are interested to investigate and optimize the process parameters involved in supercritical antisolvent process (SAS). In SAS, the substrate of interest is generally dissolved in a suitable organic solvent and subsequently precipitated out during rapid solvent removal when the organic solution is sprayed into supercritical CO2. The figure shows an example of the jet breakup at different pressures during SAS.
In our research, we have successfully encapsulated paclitaxel in Poly L lactide with sustained release. Different morphology particles were obtained when the operating conditions were varied. We are also interested in the mechanisms of jet disintegration and atomization processes involved at supercritical conditions and the implications on particle size and morphology.
Front Tracking/Finite Difference Computational Fluid Dynamics Simulation of the Electrohydrodynamic Atomization Process Liang Kuang Lim
Abstract
Electrohydrodynamic atomization process has received intense research attention recently due to its ability to generate monodisperse droplets. The process can be controlled through adjusting the electrical field strength. Recently, in addition to the electric potential applied tothe capillary nozzle, an additional ring electrode has been placed near the tip of the nozzle in order to modulate the electrical field distribution in space and to control both the spray mode and the size of droplet. To further understand the electrohydrodynamic atomization process and the effect of the secondary electrical field source, a Computational Fluid Dynamic (CFD) simulation method, the Front Tracking/Finite Difference method, has been employed to simulate the electrohydrodynamic atomization process. In this numerical method, the Navier-Stokes equation is solved for the flow of both the liquid and the ambient airflow near the nozzle tip, and the interface is monitored using a front tracking approach. At the interface, both surface tension and electrical stress due to the surface charging and applied electrical field are taken into account. The surface charging effect was modeled bya simplified constant surface charge density approach. To accurately include the effect of the secondary electrical field source due tothe ring electrode, the electrical field distribution was first calculated over a large domain. This then provided the electrical boundary conditions for the detailed CFD analysis near the region of the tip of the nozzle. The formation of the Taylor Cone, liquid jet and droplets was successfully simulated. A comparison of the results from simulations and from experiments shows that they agree reasonably well. The numerical simulation method proposed in this paper can be employed as a platform for more in depth investigation, analysis and optimization of electrohydrodynamic atomization process.
Comparison between experimental (A,B,C) and the corresponding simulated mode of spray (D,E,F). The electrical field strength near the nozzle is increased from left to right (A to C). The electrical charge densities kept constant throughout.
Velocity vectors (Left) and streamline (Right) of the liquid flow inside the Taylor Cone and Jet of the Electrohydrodynamic Atomization process.
The changes in droplet size with different ring-nozzle electrical potential differences in the Single Taylor Cone Single Jet Mode can be used to control the size of the particle fabricated.