Carbon Dioxide-Generating PLG Nanoparticles for Controlled Anti-Cancer Drug Delivery.

PURPOSE: Poly(D,L-lactide-co-glycolide) (PLG) nanoparticles containing doxorubicin and mineralized calcium carbonate were fabricated and their anti-tumor efficacy was tested using a neuroblastoma-bearing mouse model. METHODS: PLG nanoparticles were prepared by a double emulsion (water-in-oil-in-water; W/O/W) method. Calcium carbonate was mineralized within the PLG nanoparticles during the emulsion process. Rabies virus glycoprotein (RVG) peptide was chemically introduced to the surface of the PLG nanoparticles as a targeting moiety against neuroblastoma. The cytotoxicity and cellular uptake characteristics of these nanoparticles were investigated in vitro. Moreover, their therapeutic efficacy was evaluated using a tumor-bearing mouse model. RESULTS: Mineralized calcium carbonate in PLG nanoparticles was ionized at acidic pH and generated carbon dioxide gas, which resultantly accelerated the release of doxorubicin from the nanoparticles. RVG peptide-modified, gas-generating PLG nanoparticles showed a significantly enhanced targeting ability to neuroblastoma and an increased therapeutic efficacy in vivo as compared with free doxorubicin. CONCLUSIONS: Targeting ligand-modified polymer nanoparticles containing both anti-cancer drug and mineralized calcium carbonate could be useful for cancer treatment.

Optical Imaging and Gene Therapy with Neuroblastoma-Targeting Polymeric Nanoparticles for Potential Theranostic Applications.

Recently, targeted delivery systems based on functionalized polymeric nanoparticles have attracted a great deal of attention in cancer diagnosis and therapy. Specifically, as neuroblastoma occurs in infancy and childhood, targeted delivery may be critical to reduce the side effects that can occur with conventional approaches, as well as to achieve precise diagnosis and efficient therapy. Thus, biocompatible poly(d,l-lactide-co-glycolide) (PLG) nanoparticles containing an imaging probe and therapeutic gene are prepared, followed by modification with rabies virus glycoprotein (RVG) peptide for neuroblastoma-targeting delivery. RVG peptide is a well-known neuronal targeting ligand and is chemically conjugated to PLG nanoparticles without changing their size or shape. RVG-modified nanoparticles are effective in specifically targeting neuroblastoma both in vitro and in vivo. RVG-modified nanoparticles loaded with a fluorescent probe are useful to detect the tumor site in a neuroblastoma-bearing mouse model, and those encapsulating a therapeutic gene cocktail (siMyc, siBcl-2, and siVEGF) significantly suppressed tumor growth in the mouse model. This approach to designing and tailoring of polymeric nanoparticles for targeted delivery may be useful in the development of multimodality systems for theranostic approaches.

Multi-Functional Polymer Nanoparticles with Enhanced Adipocyte Uptake and Adipocytolytic Efficacy.

Multi-functional polymer nanoparticles have been widely utilized to improve cellular uptake and enhance therapeutic efficacy. In this study, it is hypothesized that the cellular uptake of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles loaded with calcium carbonate minerals into adipocytes can be improved by covalent modification with nona-arginine (R9 ) peptide. It is further hypothesized that the internalization mechanism of R9 -modified PLG nanoparticles by adipocytes may be contingent on the concentration of R9 peptide present in the nanoparticles. R9 -modified PLG nanoparticles followed the direct penetration mechanism when the concentration of R9 peptide in the nanoparticles reached 38 µM. Notably, macropinocytosis is the major endocytic mechanism when the R9 peptide concentration is ≤ 26 µM. The endocytic uptake of the nanoparticles effectively generated carbon dioxide gas at an endosomal pH, resulting in significant adipocytolytic effects in vitro, which are further supported by the findings in an obese mouse model induced by high-fat diet. Gas-generating PLG nanoparticles, modified with R9 peptide, demonstrated localized reduction of adipose tissue (reduction of 13.1%) after subcutaneous injection without significant side effects. These findings highlight the potential of multi-functional polymer nanoparticles for the development of effective and targeted fat reduction techniques, addressing both health and cosmetic considerations.

Tumor-specific cytolysis by peptide-conjugated echogenic polymer micelles.

Interest in multifunctional polymer nanoparticles for targeted delivery of anti-cancer drugs has grown significantly in recent years. In this study, tumor-targeting echogenic polymer micelles were prepared from poly(ethylene glycol) methyl ether-alkyl carbonate (mPEG-AC) derivatives, and their potential in cancer therapy was assessed. Various mPEG derivatives with carbonate linkages were synthesized via an alkyl halide reaction between mPEG and alkyl chloroformate. Micelle formation using polymer amphiphiles in aqueous media and the subsequent carbon dioxide (CO2) gas generation from the micelles was confirmed. Their ability to target neuroblastoma was substantially enhanced by incorporating the rabies virus glycoprotein (RVG) peptide. RVG-modified gas-generating micelles significantly inhibited tumor growth in a tumor-bearing mouse model owing to CO2 gas generation within tumor cells and resultant cytolytic effects, showing minimal side effects. The development of multifunctional polymer micelles may offer a promising therapeutic approach for various diseases, including cancer.

Co-delivery of vascular endothelial growth factor and angiopoietin-1 using injectable microsphere/hydrogel hybrid systems for therapeutic angiogenesis.

PURPOSE: We hypothesized that combined delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) using microsphere/hydrogel hybrid systems could enhance mature vessel formation compared with administration of each factor alone. METHODS: Hybrid delivery systems composed of alginate hydrogels and poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres containing angiogenic factors were prepared. The release behavior of angiogenic factors from hybrid systems was monitored in vitro. The hybrid systems were injected into an ischemic rodent model, and blood vessel formation at the ischemic site was evaluated. RESULTS: The sustained release over 4 weeks of both VEGF and Ang-1 from hybrid systems was achieved in vitro. Co-delivery of VEGF and Ang-1 was advantageous to retain muscle tissues and significantly induced vessel enlargement at the ischemic site, compared to mice treated with either VEGF or Ang-1 alone. CONCLUSIONS: Sustained and combined delivery of VEGF and Ang-1 significantly enhances vessel enlargement at the ischemic site, compared with sustained delivery of either factor alone. Microsphere/hydrogel hybrid systems may be a promising vehicle for delivery of multiple drugs for many therapeutic applications.

Intracellular Glucose-Depriving Polymer Micelles for Antiglycolytic Cancer Treatment.

A new anticancer strategy to exploit abnormal metabolism of cancer cells rather than to merely control the drug release or rearrange the tumor microenvironment is reported. An antiglycolytic amphiphilic polymer, designed considering the unique metabolism of cancer cells (Warburg effect) and aimed at the regulation of glucose metabolism, is synthesized through chemical conjugation between glycol chitosan (GC) and phenylboronic acid (PBA). GC-PBA derivatives form stable micellar structures under physiological conditions and respond to changes in glucose concentration. Once the micelles accumulate at the tumor site, intracellular glucose capture occurs, and the resultant energy deprivation through the inhibition of aerobic glycolysis remarkably suppresses tumor growth without significant side effects in vivo. This strategy highlights the need to develop safe and effective cancer treatment without the use of conventional anticancer drugs.

On/off switchable physical stimuli regulate the future direction of adherent cellular fate.

The utilization of cell-manipulating techniques reveals information about biological behaviors suited to address a wide range of questions in the field of life sciences. Here, we introduced an on/off switchable physical stimuli technique that offers precise stimuli for reversible cell patterning to allow regulation of the future direction of adherent cellular behavior by leveraging enzymatically degradable alginate hydrogels with defined chemistry and topography. As a proof of concept, targeted muscle cells adherent to TCP exhibited a reshaped structure when the hydrogel-based physical stimuli were applied. This simple tool offers easy manipulation of adherent cells to reshape their morphology and to influence future direction depending on the characteristics of the hydrogel without limitations of time and space. The findings from this study are broadly applicable to investigations into the relationships between cells and physiological extracellular matrix environments as well as has potential to open new horizons for regenerative medicine with manipulated cells.

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

PURPOSE: We 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. METHODS: A 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. RESULTS: The 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. CONCLUSION: A 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.

Local and sustained vascular endothelial growth factor delivery for angiogenesis using an injectable system.

PURPOSE: We hypothesize that a microsphere/hydrogel combination system could be useful for the local and sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) to enhance angiogenesis in vivo. METHODS: Poly(D,L-lactide-co-glycolide) (PLGA) microspheres containing rhVEGF were loaded into alginate gels by ionic cross-linking. The rhVEGF release from the system was monitored and bioactivity was tested in vitro. The combination system was subcutaneously injected into mice using a syringe, and new blood vessel formation was evaluated. RESULTS: Sustained rhVEGF release from the combination system was observed for 3 weeks, and the released rhVEGF remained bioactive. Endothelial cell proliferation was significantly enhanced when cells were cultured with the rhVEGF-releasing combination system in vitro. When the combination system was implanted, the granulation tissue layer was thicker with more newly formed blood vessels than that with a single dose VEGF injection. CONCLUSION: The rhVEGF release was controlled by varying relative portions of microspheres and hydrogels in combination delivery systems, which efficiently promoted new blood vessel formation in vivo. This combination system could be a promising delivery vehicle for therapeutic angiogenesis.

Characteristics of novel monofilament sutures prepared by conjugate spinning.

Compared with braided multifilament sutures, absorbable monofilaments are attractive suture materials as they exhibit less tissue drag and cause less tearing because of their smooth surfaces. However, monofilament sutures are less flexible and more difficult to tie a knot than multifilament ones, and their knots are more likely to loosen due to inferior knot security. Although various approaches have been reported to improve the flexibility of monofilament sutures, they still have limitations regarding poor knot security. To address this problem, we developed a novel technique to fabricate monofilament sutures by a conjugate spinning method, resulting in the formation of a sea/islands type of bicomponent monofilament suture. These sea/islands type bicomponent monofilament sutures, which can place many fine strands of a polymeric fiber within a matrix of another polymer, exhibited excellent knot security, flexibility, and low strain energy, compared with commercially available monofilament sutures.

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.

Tuning the sphere-to-rod transition in the self-assembly of thermoresponsive polymer hybrids.

Nano-scale drug delivery systems have undergone extensive development, and control of size and structure is critical for regulation of their biological responses and therapeutic efficacy. Amphiphilic polymers that form self-assembled structures in aqueous media have been investigated and used for the diagnosis and therapy of various diseases, including cancer. Here, we report the design and fabrication of thermoresponsive polymeric micelles from alginate conjugated with poly(N-isopropylacrylamide) (PNIPAAm). Alginate-PNIPAAm hybrids formed self-aggregated structures in response to temperature changes near body temperature. A structural transition from micellar spheres to rods of alginate-PNIPAAm hybrids was observed depending on the molecular weight of PNIPAAm and the polymer concentration. Additionally, hydrogels with nanofibrous structures were formed by simply increasing the polymer concentration. This approach to controlling the structure of polymer micelles from nanoparticles to fibrous hydrogels may be useful in applications in drug delivery and tissue engineering.

Controlled degradation of hydrogels using multi-functional cross-linking molecules.

Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the degradation rates and mechanical stiffness of gels than bi-functional cross-linking molecules. To address the possibility, we isolated alpha-L-guluronate residues of sodium alginate, and oxidized them to prepare poly(aldehyde guluronate) (PAG). Hydrogels were formed with either poly(acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linking molecule or adipic acid dihydrazide (AAD) as a bi-functional cross-linking molecule. The initial properties and degradation behavior of both PAG gel types were monitored. PAG/PAH hydrogels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels, at the same concentration of cross-linking functional groups. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel at the same concentration of functional groups. This approach to regulating gel properties with multifunctional cross-linking molecules could be broadly used in hydrogels.

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.

The height of cell-adhesive nanoposts generated by block copolymer/surfactant complex systems influences the preosteoblast phenotype.

In tissue engineering, the nanoscale topography of the substrate is important, because transplanted cells can recognize and respond to topographical patterns, allowing control of gene expression and tissue formation. In this study, we hypothesized that the height of cell-adhesive nanoposts could regulate cell phenotype. Nano-patterned surfaces were generated via self-assembly of polystyrene-b-poly(ethylene oxide)/dodecylbenzenesulfonic acid (PS-b-PEO/DBSA) complex systems. The height of PS nanoposts, which are considered to be cell-adhesion domains, was varied from 11 to 43 nm, while nanopost size and the center-to-center distance between nanoposts were kept constant. Adhesion, growth, and differentiation of mouse preosteoblasts (MC3T3-E1) cultured on the nano-patterned surfaces were significantly influenced by nanopost height. This approach therefore holds great promise for the design of biomedical devices, as well as tissue engineering scaffolds.

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.

Injectable microsphere/hydrogel hybrid system containing heat shock protein as therapy in a murine myocardial infarction model.

Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.

Arginine-engrafted biodegradable polymer for the systemic delivery of therapeutic siRNA.

Small interfering RNA (siRNA) represent an interesting class of developmental nucleic acid-based therapeutics. Cationic carriers for deoxyribonucleic acids (DNA) are potential vehicles for siRNA delivery. However, in contrast to supercoiled plasmid DNA, the physical properties of siRNA molecules induces the formation of larger, loosely-packed complexes with most polycationic carriers, and consequently, poor target silencing. Here, we investigate a gene delivery agent, arginine-grafted bioreducible poly (disulfide amine) polymer (ABP) for siRNA delivery as it contains arginine residues with siRNA binding properties. ABP combines the attributes of polycations and poly disulfide-amines namely- excellent cell-penetrability and rapid release after disulphide bond reduction in the intracellular compartment. ABP bound siRNA, assembled into stable 150 nm sized nanoparticles and efficiently released complexed siRNA upon cellular entry. We investigated the utility of ABP in a combinatorial RNAi strategy for solid cancer therapy. Systemic administration of ABP-siRNA resulted in a preferential and enhanced accumulation of carrier-siRNA complexes in the tumor tissue. Two administrations of the formulation with a siRNA cocktail targeting Bcl-2, VEGF and Myc at 0.3 mg total siRNA/kg body weight could effectively regress advanced stage tumors. Our results establish the promise of ABP as a common systemic delivery platform for both siRNA and DNA therapeutics.

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.

Hydrogel-based biomimetic environment for in vitro modulation of branching morphogenesis.

The mechanical properties of the cellular microenvironment dramatically alter during tissue development and growth. Growing evidence suggests that physical microenvironments and mechanical stresses direct cell fate in developing tissue. However, how these physical cues affect the tissue morphogenesis remains a major unknown. We explain here that the physical properties of the cell and tissue microenvironment, biomimetically reproduced by using hydrogel, guide the tissue morphogenesis in the developmental submandibular gland (SMG). In particular, the softer gel enhances the bud expansion and cleft formation of SMG, whereas the stiffer gel attenuates them. These morphological changes in SMG tissue are led by soluble factors (FGF7/10) induction regulated by cell traction force derived from the tissue deformation. Our findings suggest that cells sense the mechanics of their surrounding environment and alter their properties for self-organization and the following tissue morphogenesis. Also, physically designed hydrogel material is a valuable tool for producing the biomimetic microenvironment to explore how physical cues affect tissue morphogenesis and to modulate tissue morphogenesis for in vitro tissue synthesis.