Jangwook Lee

Facile control of porous structures of polymer microspheres using an osmotic agent for pulmonary delivery.

It has been challenging to prepare polymeric microspheres with controlled porous structures for many biomedical applications, particularly for pulmonary drug delivery. Here, we report the use of bovine serum albumin (BSA) as an osmotic agent in order to control the porous structure of poly(D,L-lactide-co-glycolide) (PLGA) microspheres prepared by a double emulsion method. BSA was useful to induce osmosis between internal and external water phases during the double emulsion process, resulting in the fabrication of microspheres with controllable, uniform porous structures. The pore size of PLGA microspheres was controlled independently from the particle size by this approach. The use of BSA as an osmotic agent reduced the initial burst of model proteins (e.g., insulin and VEGF) entrapped in the porous microspheres, and the sustained release of VEGF was achieved for two weeks in vitro. This approach to controlling porous structures of polymer microspheres could be useful to develop novel pulmonary drug delivery systems.

Controlled delivery of heat shock protein using an injectable microsphere/hydrogel combination system for the treatment of myocardial infarction

Myocardial infarction causes a high rate of morbidity and mortality worldwide, and heat shock proteins as molecular chaperones have been attractive targets for protecting cardiomyoblasts under environmental stimuli. In this study, in order to enhance the penetration of heat shock protein 27 (HSP27) across cell membranes, we fused HSP27 with transcriptional activator (TAT) derived from human immunodeficiency virus (HIV) as a protein transduction domain (PTD). We loaded the fusion protein (TAT-HSP27) into microsphere/hydrogel combination delivery systems to control the release behavior for prolonged time periods. We found that the release behavior of TAT-HSP27 was able to be controlled by varying the ratio of PLGA microspheres and alginate hydrogels. Indeed, the released fusion protein maintained its bioactivity and could recover the proliferation of cardiomyoblasts cultured under hypoxic conditions. This approach to controlling the release behavior of TAT-HSP27 using microsphere/hydrogel combination delivery systems may be useful for treating myocardial infarction in a minimally invasive manner.

Preparation of budesonide-loaded porous PLGA microparticles and their therapeutic efficacy in a murine asthma model

Inhaling corticosteroids, such as budesonide (BD), is the most common treatment for asthma. However, frequent steroid administration is associated with many side effects. We hypothesized that porous microparticles containing BD could provide an effective treatment method for asthma, as the sustained delivery of corticosteroid and a reduced number of doses could be achieved using porous polymeric microparticles. Porous microparticles were prepared from poly(lactic-co-glycolic acid) (PLGA) by a water-in-oil-in-water double emulsion method with ammonium bicarbonate as the porogen. Varying the porogen concentration controlled the morphology, particle size, and pore size of the PLGA microparticles, with particle size and pore size increasing as the porogen concentration increased. The BD loading efficiency in the porous PLGA microparticles was about 60%, and BD was released from the porous microparticles in a sustained manner for 24h in vitro. Lung uptake efficiency of the porous PLGA microparticles in mice was significantly higher than that of non-porous PLGA microparticles. Budesonide-loaded porous PLGA microparticles were delivered to asthmatic mice, and the numbers of inflammatory cells in bronchoalveolar lavage (BAL) fluid and tissue sections were significantly reduced when the drug was administrated every 3days. We also found significantly reduced bronchial hyperresponsiveness of asthmatic mice after treatment with budesonide-loaded porous PLGA microparticles. This approach to controlling the porous structure of polymeric microparticles, as well as the release behavior of drugs from the microparticles, could have useful applications in the pulmonary delivery of many therapeutic drugs.

T cell-specific siRNA delivery using antibody-conjugated chitosan nanoparticles.

The intracellular delivery of small interfering RNA (siRNA) plays a key role in RNA interference (RNAi) and provides an emerging technique to treat various diseases, including infectious diseases. Chitosan has frequently been used in gene delivery applications, including siRNA delivery. However, studies regarding the modification of chitosan with antibodies specifically targeting T cells are lacking. We hypothesized that chitosan nanoparticles modified with T cell-specific antibodies would be useful for delivering siRNA to T cells. CD7-specific single-chain antibody (scFvCD7) was chemically conjugated to chitosan by carbodiimide chemistry, and nanoparticles were prepared by a complex coacervation method in the presence of siRNA. The mean diameter and zeta potential of the scFvCD7-chitosan/siRNA nanoparticles were approximately 320 nm and +17 mV, respectively, and were not significantly influenced by the coupling of antibody to chitosan. The cellular association of antibody-conjugated nanoparticles to CD4+ T cell lines as well as gene silencing efficiency in the cells was significantly improved compared to nonmodified chitosan nanoparticles. This approach to introducing T cell-specific antibody to chitosan nanoparticles may find useful applications for the treatment of various infectious diseases.

Active blood vessel formation in the ischemic hindlimb mouse model using a microsphere/hydrogel combination system.

PurposeWe hypothesize that the controlled delivery of rhVEGF using a microsphere/hydrogel combination system could be useful to achieve active blood vessel formation in the ischemic hindlimb mouse model, which is clinically relevant for therapeutic angiogenesis without multiple administrations.MethodsA combination of poly(d,l-lactide-co-glycolide) (PLGA) microspheres and alginate hydrogels containing rhVEGF was prepared and injected intramuscularly into the ischemic hindlimb site of mouse model, and new blood vessel formation near the ischemic site was evaluated.ResultsThe controlled release of rhVEGF from the combination system effectively protected muscles in ischemic regions from tissue necrosis. Interestingly, the number of newly formed, active blood vessels was significantly increased in mice treated with the rhVEGF-releasing combination system.ConclusionA microsphere/hydrogel combination system provided a useful means to deliver therapeutic angiogenic molecules into the body for the treatment of ischemic vascular diseases, which could reduce the number of administrations of many types of drugs.

Injectable microsphere/hydrogel combination systems for localized protein delivery.

Injectable delivery systems for therapeutic proteins (e.g., hydrogels and microspheres) have attracted wide attention. Hydrogels, however, may release their hydrophilic contents too rapidly in a large initial burst, and phagocytes may clear microspheres within a relatively short time period after administration. We hypothesized that microsphere/hydrogel combination systems could achieve a controlled and sustained release of proteins as an injectable delivery system. To test this hypothesis, we prepared PLGA microspheres containing a model protein and mixed these with alginate gels. The mixing ratio of the components was the primary controlling parameter of the protein release. This approach could be useful for development of injectable and localized drug delivery systems.

Preparation and characterization of nonaarginine-modified chitosan nanoparticles for siRNA delivery

Chitosan-based nanoparticles have been widely used as a carrier for gene delivery due to their low toxicity and the positively charged amino groups in chitosan. In this study, we hypothesized that introduction of nonaarginine to chitosan could improve its ability to form a complex with siRNA, as well as enhance the cellular uptake and transfection efficiency of chitosan-based nanoparticles. To this end, a peptide with nine repeating arginine residues was chemically coupled to the chitosan backbone, and various characteristics of nonaarginine-chitosan/siRNA nanoparticles were investigated. The mean diameter and zeta potential of the nonaarginine-chitosan/siRNA nanoparticles were dependent on the amount of nonaarginine conjugated to chitosan. Nonaarginine-modified chitosan/siRNA nanoparticles demonstrated enhanced cellular association and transfection efficiency in vitro, while maintaining a low level of cytotoxicity. In conclusion, nonaarginine-modified chitosan should be considered a potential carrier for various gene delivery applications.

Sequential delivery of TAT-HSP27 and VEGF using microsphere/hydrogel hybrid systems for therapeutic angiogenesis.

Ischemic disease is associated with high mortality and morbidity rates, and therapeutic angiogenesis via systemic or local delivery of protein drugs is one potential approach to treat the disease. In this study, we hypothesized that combined delivery of TAT-HSP27 (HSP27 fused with transcriptional activator) and VEGF could enhance the therapeutic efficacy in an ischemic mouse model, and that sequential release could be critical in therapeutic angiogenesis. Alginate hydrogels containing TAT-HSP27 as an anti-apoptotic agent were prepared, and porous PLGA microspheres loaded with VEGF as an angiogenic agent were incorporated into the hydrogels to prepare microsphere/hydrogel hybrid delivery systems. Sequential in vitro release of TAT-HSP27 and VEGF was achieved by the hybrid systems. TAT-HSP27 was depleted from alginate gels in 7 days, while VEGF was continually released for 28 days. The release rate of VEGF was attenuated by varying the porous structures of PLGA microspheres. Sequential delivery of TAT-HSP27 and VEGF was critical to protect against muscle degeneration and fibrosis, as well as to promote new blood vessel formation in the ischemic site of a mouse model. This approach to controlling the sequential release behaviors of multiple drugs could be useful in the design of novel drug delivery systems for therapeutic angiogenesis.

Doxorubicin-loaded alginate-g-poly(N-isopropylacrylamide) micelles for cancer imaging and therapy.

Chemotherapy is a widely adopted method for the treatment of cancer. However, its use is often limited due to side effects produced by anti-cancer drugs. Therefore, various drug carriers, including polymeric micelles, have been investigated to find a method to overcome this limitation. In this study, alginate-based, self-assembled polymeric micelles were designed and prepared using alginate-g-poly(N-isopropylacrylamide) (PNIPAAm). Amino-PNIPAAm was chemically introduced to the alginate backbone via carbodiimide chemistry. The resulting polymer was dissolved in distilled water at room temperature and formed self-assembled micelles at 37 °C. Characteristics of alginate-g-PNIPAAm micelles were dependent on the molecular weight of PNIPAAm, the degree of substitution, and the polymer concentration. Doxorubicin (DOX), a model anti-cancer drug, was efficiently encapsulated in alginate-g-PNIPAAm micelles, and sustained release of DOX from the micelles was achieved at 37 °C in vitro. These micelles accumulated at the tumor site of a tumor-bearing mouse model as a result of the enhanced permeability and retention effect. Interestingly, DOX-loaded alginate-g-PNIPAAm micelles showed excellent anti-cancer therapeutic efficacy in a mouse model without any significant side effects. This approach to designing and tailoring natural polymer-based systems to fabricate nanoparticles at human body temperature may provide a useful means for cancer imaging and therapy.

Theranostic gas-generating nanoparticles for targeted ultrasound imaging and treatment of neuroblastoma.

The development of safe and efficient diagnostic/therapeutic agents for treating cancer in clinics remains challenging due to the potential toxicity of conventional agents. Although the annual incidence of neuroblastoma is not that high, the disease mainly occurs in children, a population vulnerable to toxic contrast agents and therapeutics. We demonstrate here that cancer-targeting, gas-generating polymeric nanoparticles are useful as a theranostic tool for ultrasound (US) imaging and treating neuroblastoma. We encapsulated calcium carbonate using poly(d,l-lactide-co-glycolide) and created gas-generating polymer nanoparticles (GNPs). These nanoparticles release carbon dioxide bubbles under acidic conditions and enhance US signals. When GNPs are modified using rabies virus glycoprotein (RVG) peptide, a targeting moiety to neuroblastoma, RVG-GNPs effectively accumulate at the tumor site and substantially enhance US signals in a tumor-bearing mouse model. Intravenous administration of RVG-GNPs also reduces tumor growth in the mouse model without the use of conventional therapeutic agents. This approach to developing theranostic agents with disease-targeting ability may provide useful strategy for the detection and treatment of cancers, allowing safe and efficient clinical applications with fewer side effects than may occur with conventional agents.