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1.  Osteochondral Tissue Regeneration using a Bilayered Composite Hydrogel with Modulating Dual Growth Factor Release Kinetics in a Rabbit Model 
Biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) composite hydrogels have been investigated for the delivery of growth factors (GFs) with the aid of gelatin microparticles (GMPs) and stem cell populations for osteochondral tissue regeneration. In this study, a bilayered OPF composite hydrogel that mimics the distinctive hierarchical structure of native osteochondral tissue was utilized to investigate the effect of transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) on osteochondral tissue regeneration in a rabbit full-thickness osteochondral defect model. The four groups investigated included (i) a blank control (no GFs), (ii) GMP-loaded IGF-1 alone, (iii) GMP-loaded IGF-1 and gel-loaded TGF-β3, and (iv) GMP-loaded IGF-1 and GMP-loaded TGF-β3 in OPF composite hydrogels. The results of an in vitro release study demonstrated that TGF-β3 release kinetics could be modulated by the GF incorporation method. At 12 weeks post-implantation, the quality of tissue repair in both chondral and subchondral layers was analyzed based on quantitative histological scoring. All groups incorporating GFs resulted in a significant improvement in cartilage morphology compared to the control. Single delivery of IGF-1 showed higher scores in subchondral bone morphology as well as chondrocyte and glycosaminoglycan amount in adjacent cartilage tissue when compared to a dual delivery of IGF-1 and TGF-β3, independent of the TGF-β3 release kinetics. The results suggest that although the dual delivery of TGF-β3 and IGF-1 may not synergistically enhance the quality of engineered tissue, the delivery of IGF-1 alone from bilayered composite hydrogels positively affects osteochondral tissue repair and holds promise for osteochondral tissue engineering applications.
PMCID: PMC3661728  PMID: 23541928
Hydrogel; osteochondral defect; transforming growth factor-β3; insulin-like growth factor-1
2.  Impact of flow pulsatility on arterial drug distribution in stent-based therapy 
Drug-eluting stents reside in a dynamic fluid environment where the extent to which drugs are distributed within the arterial wall is critically modulated by the blood flowing through the arterial lumen. Yet several factors associated with the pulsatile nature of blood flow and their impact on arterial drug deposition has not been fully investigated. We employed an integrated framework comprising bench-top and computational models to explore the factors governing the time-varying fluid dynamic environment within the vasculature and their effects on arterial drug distribution patterns. A custom-designed bench-top framework comprising a model of a single drug-eluting stent strut and a poly-vinyl alcohol-based hydrogel as a model tissue bed simulated fluid flow and drug transport under fully apposed strut settings. Bench-top experiments revealed a relative independence between drug distribution and the factors governing pulsatile flow and these findings were validated with the in silico model. Interestingly, computational models simulating suboptimal deployment settings revealed a complex interplay between arterial drug distribution, Womersley number and the extent of malapposition. In particular, for a stent strut offset from the wall, total drug deposition was sensitive to changes in the pulsatile flow environment, with this dependence increasing with greater wall displacement. Our results indicate that factors governing pulsatile luminal flow on arterial drug deposition should be carefully considered in conjunction with device deployment settings for better utilization of drug-eluting stent therapy for various arterial flow regimes.
PMCID: PMC3697861  PMID: 23541929
Drug-Eluting Stents; Pulsatile Blood Flow; Pharmacokinetics; Computational Fluid Dynamics
3.  Mesoporous silica-supported lipid bilayers (protocells) for DNA cargo delivery to the spinal cord 
Amorphous mesoporous silica nanoparticles (‘protocells’) that support surface lipid bilayers recently characterized in vitro as carrier constructs for small drug and DNA delivery are reported here as highly biocompatible both in vitro and in vivo, involving the brain and spinal cord following spinal delivery into the lumbosacral subarachnoid space (intrathecal; i.t.). Specifically, positively charged, 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) -cholesterol (DOTAP:Chol) liposome-formulated protocells revealed stable in vitro cargo release kinetics and cellular interleukin-10 (IL-10) transgene transfection. Recent approaches using synthetic non-viral vector platforms to deliver the pain-suppressive therapeutic transgene, IL-10, to the spinal subarachnoid space has yielded promising results in animal models of peripheral neuropathy, a condition involving aberrant neuronal communication within sensory pathways in the nervous system. Non-viral drug and gene delivery protocell platforms offer potential flexibility because cargo release-rates can be pH-dependent. We report here that i.t. delivery of protocells, with modified chemistry supporting a surface coating of DOTAP:Chol liposomes and containing the IL-10 transgene, results in functional suppression of pain-related behavior in rats for extended periods. This study is the first demonstration that protocell vectors offer amenable and enduring in vivo biological characteristics that can be applied to spinal gene delivery.
PMCID: PMC4013798  PMID: 23517784
4.  PEGylation of interferon α2 improves lymphatic exposure after subcutaneous and intravenous administration and improves antitumour efficacy against lymphatic breast cancer metastases 
The efficacy of protein-based therapeutics with indications in the treatment of lymphatic diseases is expected to be improved by enhancing lymphatic disposition. This study was therefore aimed at examining whether PEGylation can usefully be applied to improve the lymphatic uptake of interferon α2 and whether this ultimately translates into improved therapeutic efficacy against lymph-resident cancer. The lymphatic pharmacokinetics of interferon α2b (IFN, 19 kDa) and PEGylated interferon α2b (IFN-PEG12, 31 kDa) or α2a (IFN-PEG40, 60 kDa) was examined in thoracic lymph duct cannulated rats. IFN was poorly absorbed from the SC injection site (Fabs 36%) and showed little uptake into lymph after SC or IV administration (≤1%). In contrast, IFN-PEG12 was efficiently absorbed from the SC injection site (Fabs 82%) and approximately 20% and 8% of the injected dose was recovered in thoracic lymph over 30 hours after SC or IV administration respectively. IFN-PEG40, however, was incompletely absorbed from the SC injection site (Fabs 23%) and showed similar lymphatic access after SC administration to IFN-PEG12 (21%). The recovery of IFN-PEG40 in thoracic lymph after IV administration, however, was significantly greater (29%) when compared to IV IFN-PEG12. The anti-tumour efficacy of interferon against axillary metastases of a highly lymph-metastatic variant of human breast MDA-MB-231 carcinoma was significantly increased by SC administration of lymph-targeted IFN-PEG12 when compared to the administration of IFN on the ipsilateral side to the axillary metastasis. Optimal PEGylation may therefore represent a viable approach to improving the lymphatic disposition and efficacy of therapeutic proteins against lymphatic diseases.
PMCID: PMC4022972  PMID: 23499718
PEGylation; interferon; metastasis; pharmacokinetics; lymphatic
5.  Dexamethasone-poly(dimethylamino)ethyl methacrylate (pDMAEMA) conjugates reduce inflammatory biomarkers in human intestinal epithelial monolayers 
The mucoadhesive polymer, poly(dimethylamino)ethyl methacrylate (pDMAEMA) was synthesised by living radical polymerisation and subsequently conjugated by esterification to the anti-inflammatory corticosteroid, dexamethasone, to separately yield two concentrations of conjugates with ratios of 10:1 and 20:1 active:polymer. The hypothesis was to test whether the active agent maintained in vitro bioactivity when exposed to the apical side of human intestinal epithelial monolayers, Caco-2 and mucous-covered HT29-MTX-E12 (E12). HPLC analysis showed that 80% of the dexamethasone in both conjugates was attached to pDMAEMA. Similar to pDMAEMA, fluorescently-labelled dexamethasone-pDMAEMA conjugates were bioadhesive to Caco-2 and mucoadhesive to E12. Apical addition of conjugates suppressed mRNA expression of the inflammatory markers, NURR1 and ICAM-1 in E12 following stimulation by PGE2 and TNF-α, respectively. Conjugates also suppressed TNF-α stimulated cytokine secretion to the basolateral side of Caco-2 monolayers. pDMAEMA was inactive in these assays. Measurement of dexamethasone permeability from conjugates across monolayers suggested that conjugation reduced permeability compared to free dexamethasone. LDH assay indicated that conjugates were not cytotoxic to monolayers at high concentrations. Anti-inflammatory agents can therefore be successfully conjugated to polymers and they retain adhesion and bioactivity to enable formulation for topical administration.
PMCID: PMC4047761  PMID: 19110018
6.  In vivo Targeting of Adoptively Transferred T-cells with Antibody- and Cytokine-Conjugated Liposomes 
In adoptive cell therapy (ACT), autologous tumor-specific T-cells isolated from cancer patients are activated and expanded ex vivo, then infused back into the individual to eliminate metastatic tumors. A major limitation of this promising approach is the rapid loss of ACT T-cell effector function in vivo due to the highly immunosuppressive environment in tumors. Protection of T-cells from immunosuppressive signals can be achieved by systemic administration of supporting adjuvant drugs such as interleukins, chemotherapy, and other immunomodulators, but these adjuvant treatments are often accompanied by serious toxicities and may still fail to optimally stimulate lymphocytes in all tumor and lymphoid compartments. Here we propose a novel strategy to repeatedly stimulate or track ACT T-cells, using cytokines or ACT-cell-specific antibodies as ligands to target PEGylated liposomes to transferred T-cells in vivo. Using F(ab′)2 fragments against a unique cell surface antigen on ACT cells (Thy1.1) or an engineered interleukin-2 (IL-2) molecule on an Fc framework as targeting ligands, we demonstrate that >95% of ACT cells can be conjugated with liposomes following a single injection in vivo. Further, we show that IL-2-conjugated liposomes both target ACT cells and are capable of inducing repeated waves of ACT T-cell proliferation in tumor-bearing mice. These results demonstrate the feasibility of repeated functional targeting of T-cells in vivo, which will enable delivery of imaging contrast agents, immunomodulators, or chemotherapy agents in adoptive cell therapy regimens.
PMCID: PMC4046900  PMID: 23770010
immunoliposomes; interleukin-2 (IL-2); cancer immunotherapy; adoptive cell therapy; targeted delivery; melanoma
7.  A Simple Way to Enhance Doxil® Therapy: Drug Release from Liposomes at the Tumor Site by Amphiphilic Block Copolymer 
The antitumor efficacy of Doxil® is hindered by the poor release of the active drug from the liposome at the tumor sites. This study investigates a possibility to enhance drug release from the liposomes and increase therapeutic efficacy of Doxil® by administering Pluronic block copolymers once the liposomal drug accumulates in the tumor sites. In our study, the fluorescence de-quenching experiments were designed to investigate the drug release from liposome by Pluronic P85. MTT cytotoxicity assay and confocal microscopy images were carried out to determine whether Pluronic P85 could facilitate release of Dox from Doxil®. Anti-tumor growth and distribution of drug were evaluated when Pluronic P85 was injected 1 hr, 48 hrs, or 96 hrs after the Doxil® administration in A2780 human ovarian cancer xenografts. Addition of Pluronic P85 resulted in release of Dox from the liposomes accompanied with significant increases of Dox delivery and cytotoxic effect in cancer cells. The greatest anti-tumor effect of single injection of Doxil® was achieved when Pluronic P85 was administered 48 hrs after Doxil®. The Confocal tile scanning images of tumor section showed that copolymer treatment induced the release of the drug in the tumors from the vessels regions to the bulk of the tumor. No release of the drug remaining in circulation was observed. Our study has demonstrated a simple approach for localized release of Dox from liposome by Pluronic P85 at the tumor site, which was therapeutically beneficial.
PMCID: PMC3661699  PMID: 23474033
Liposome; Doxorubicin; copolymer; combination; cancer
8.  Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers 
Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5 kg mol−1 methoxy-terminated PEG corona. While a 5 kg mol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8 kg mol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associate with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This characterization of nanocarrier uptake and targeting provides promise for optimizing drug delivery to macrophages for TB treatment and establishes a general route for optimizing targeted formulations of nanocarriers for specific delivery at targeted sites.
PMCID: PMC3662248  PMID: 23419950
9.  Photodynamic Nanomedicine in the Treatment of Solid Tumors: Perspectives and Challenges 
Photodynamic therapy (PDT) is a promising treatment strategy where activation of photosensitizer drugs with specific wavelengths of light results in energy transfer cascades that ultimately yield cytotoxic reactive oxygen species which can render apoptotic and necrotic cell death. Without light the photosensitizer drugs are minimally toxic and the photoactivating light itself is non-ionizing. Therefore, harnessing this mechanism in tumors provides a safe and novel way to selectively eradicate tumor with reduced systemic toxicity and side effects on healthy tissues. For successful PDT of solid tumors, it is necessary to ensure tumor-selective delivery of the photosensitizers, as well as, the photoactivating light and to establish dosimetric correlation of light and drug parameters to PDT-induced tumor response. To this end, the nanomedicine approach provides a promising way towards enhanced control of photosensitizer biodistribution and tumor-selective delivery. In addition, refinement of nanoparticle designs can also allow incorporation of imaging agents, light delivery components and dosimetric components. This review aims at describing the current state-of-the-art regarding nanomedicine strategies in PDT, with a comprehensive narrative of the research that has been carried out in vitro and in vivo, with a discussion of the nanoformulation design aspects and a perspective on the promise and challenges of PDT regarding successful translation into clinical application.
PMCID: PMC3780355  PMID: 23474028
nanomedicine; drug delivery; photodynamic therapy; cancer; targeted nanoparticles
10.  Characterizing EPR-Mediated Passive Drug Targeting using Contrast-Enhanced Functional Ultrasound Imaging 
The Enhanced Permeability and Retention (EPR) effect is extensively used in drug delivery research. Taking into account that EPR is a highly variable phenomenon, we have here set out to evaluate if contrast-enhanced functional ultrasound (ceUS) imaging can be employed to characterize EPR-mediated passive drug targeting to tumors. Using standard fluorescence molecular tomography (FMT) and two different protocols for hybrid computed tomography-fluorescence molecular tomography (CT-FMT), the tumor accumulation of a ~10 nm-sized near-infrared-fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) was evaluated in CT26 tumor-bearing mice. In the same set of animals, two different ceUS techniques (2D MIOT and 3D B-mode imaging) were employed to assess tumor vascularization. Subsequently, the degree of tumor vascularization was correlated with the degree of EPR-mediated drug targeting. Depending on the optical imaging protocol used, the tumor accumulation of the polymeric drug carrier ranged from 5-12% of the injected dose. The degree of tumor vascularization, determined using ceUS, varied from 4-11%. For both hybrid CT-FMT protocols, a good correlation between the degree of tumor vascularization and the degree of tumor accumulation was observed, with in the case of reconstructed CT-FMT, correlation coefficients of ~0.8 and p-values of <0.02. These findings indicate that ceUS can be used to characterize and predict EPR, and potentially also to pre-selecting patients likely to respond to passively tumor-targeted nanomedicine treatments.
PMCID: PMC4031451  PMID: 24631862
Drug targeting; Nanomedicine; Theranostics; Cancer; EPR; HPMA; US; FMT; CT
11.  pHLIP®-Mediated Delivery of PEGylated Liposomes to Cancer Cells 
We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, “fusogenic” pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells.
PMCID: PMC3630259  PMID: 23416366
acidosis; membrane-associated folding; peptide; fusion; endocytosis; delivery of payload; ceramide; pH-sensitive
12.  Pancreatic Cancer-Associated Cathepsin E as a Drug Activator 
Pancreatic ductal adenocarcinoma (PDAC) is challenging to treat, and better means to detect and/or treat pancreatic cancer are urgently needed to save lives. Cathepsin E (Cath E) is a proteolytic enzyme highly expressed in PDAC. In this study, a novel approach using Cath E activation of a Cath E-specific prodrug was demonstrated. Specific activation of the prodrug is expected to kill pancreatic cancer cells without harming normal pancreatic cells. A novel 5-aminolevulinic acid (5-ALA) prodrug was custom-designed to be activated selectively by endogenous Cath E within the PDAC cells. The 5-ALA prodrug was incubated with Cath E-positive and -negative tumor cells and illuminated with various doses of light. In addition, mice genetically engineered to develop PDAC were injected intravenously with the 5-ALA prodrug, and the pancreas was treated with light irradiation. One day after treatment, PDAC tissue was assessed for apoptosis. The 5-ALA prodrug was activated within the Cath E-positive tumor but not in the normal pancreatic tissue. When used in combination with light treatment, it allowed delivery of selective photodynamic therapy (PDT) to the cancerous tissues, with minimal harm to the adjacent normal tissues. With this novel Cath E activation approach, it is possible to detect pancreatic cancer cells accurately and specifically impair their viability, while sparing normal cells. This treatment could result in fewer side effects than the non-specific treatments currently in use. Cath E is a specific and effective drug activator for PDAC treatment.
PMCID: PMC3638719  PMID: 23422726
Pancreatic cancer; Drug Activation; Cathepsin E; Photodynamic Therapy; Prodrug; 5-Aminolevulinic acid
13.  Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression 
RNA interference is a promising strategy for treatment of Huntington’s disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood-brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients. Focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48 hrs following treatment with siRNA, the right (treated) and left (control) striatum was dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain.
PMCID: PMC4010143  PMID: 22921802
Focused ultrasound; siRNA; drug delivery; Huntington’s disease; blood-brain barrier
14.  Particle Shape: A New Design Parameter for Micro- and Nanoscale Drug Delivery Carriers 
Encapsulation of therapeutic agents in polymer particles has been successfully used in the development of new drug carriers. A number of design parameters that govern the functional behavior of carriers, including the choice of polymer, particle size and surface chemistry, have been tuned to optimize their performance in vivo. However, particle shape, which may also have a strong impact on carrier performance, has not been investigated. This is perhaps due to the limited availability of techniques to produce non-spherical polymer particles. In recent years, a number of reports have emerged to directly address this bottleneck and initial studies have indeed confirmed that particle shape can significantly impact the performance of polymer drug carriers. This article provides a review of this field with respect to methods of particle preparation and the role of particle shape in drug delivery.
PMCID: PMC4009069  PMID: 17544538
Nanoparticle; Nanotechnology; Morphology; Drug Delivery; Biomaterials
15.  Design and physicochemical characterisation of novel dissolving polymeric microneedle arrays for transdermal delivery of high dose, low molecular weight drugs 
Journal of Controlled Release  2014;180(100):71-80.
We describe formulation and evaluation of novel dissolving polymeric microneedle (MN) arrays for the facilitated delivery of low molecular weight, high dose drugs. Ibuprofen sodium was used as the model here and was successfully formulated at approximately 50% w/w in the dry state using the copolymer poly(methylvinylether/maleic acid). These MNs were robust and effectively penetrated skin in vitro, dissolving rapidly to deliver the incorporated drug. The delivery of 1.5 mg ibuprofen sodium, the theoretical mass of ibuprofen sodium contained within the dry MN alone, was vastly exceeded, indicating extensive delivery of the drug loaded into the baseplates. Indeed in in vitro transdermal delivery studies, approximately 33 mg (90%) of the drug initially loaded into the arrays was delivered over 24 h. Iontophoresis produced no meaningful increase in delivery. Biocompatibility studies and in vivo rat skin tolerance experiments raised no concerns. The blood plasma ibuprofen sodium concentrations achieved in rats (263 μg ml− 1 at the 24 h time point) were approximately 20 times greater than the human therapeutic plasma level. By simplistic extrapolation of average weights from rats to humans, a MN patch design of no greater than 10 cm2 could cautiously be estimated to deliver therapeutically-relevant concentrations of ibuprofen sodium in humans. This work, therefore, represents a significant progression in exploitation of MN for successful transdermal delivery of a much wider range of drugs.
Graphical abstract
PMCID: PMC4034161  PMID: 24556420
Microneedles; Transdermal; Ibuprofen; Biocompatibility
16.  Application of activated nucleoside analogs for the treatment of drug-resistant tumors by oral delivery of nanogel-drug conjugates 
A majority of nanoencapsulated drugs that have shown promise in cancer chemotherapy are administered intravenously. Development of effective oral nanoformulations presents a very challenging medical goal. Here, we describe successful applications of innovative polymeric nanogels in the form of conjugates with activated nucleoside analogs for oral administration in cancer chemotherapy. Previously, we reported the synthesis of amphiphilic polyvinyl alcohol and dextrin-based nanogel conjugates with the phosphorylated 5-FU nucleoside Floxuridine and demonstrated their enhanced activity against regular and drug-resistant cancers[1]. In this study, we synthesized and evaluated oral applications of nanogel conjugates of a protected Gemcitabine, the drug never used in oral therapies. These conjugates were able to quickly release an active form of the drug (Gemcitabine 5′-mono-, di- and triphosphates) by specific enzymatic activities, or slowly during hydrolysis. Gemcitabine conjugates demonstrated up to 127 times higher in vitro efficacy than the free drug against various cancer cells, including the lines resistant to nucleoside analogs. Surprisingly, these nanogel-drug conjugates were relatively stable in gastric conditions and able to actively penetrate through the gastrointestinal barrier based on permeability studies in Caco-2 cell model. In tumor xenograft models of several drug-resistant human cancers, we observed an efficient inhibition of tumor growth and extended the life-span of the animals by 4 times that of the control with orally treated Gemcitabine- or Floxuridine-nanogel conjugates. Thus, we have demonstrated a potential of therapeutic nanogel conjugates with the activated and stabilized Gemcitabine as a successful oral drug form against Gemcitabine-resistant and other drug-resistant tumors.
PMCID: PMC3612132  PMID: 23385032
Nanogel-drug conjugates; Gemcitabine; phosphorylated nucleoside analogs; oral drug administration; drug-resistant animal tumor xenografts
17.  Liposomal Fasudil, a Rho-Kinase Inhibitor, for Prolonged Pulmonary Preferential Vasodilation in Pulmonary Arterial Hypertension 
Current pharmacological interventions for pulmonary arterial hypertension (PAH) require continuous infusions, multiple inhalations, or oral administration of drugs that act on various pathways involved in the pathogenesis of PAH. However, invasive methods of administration, short duration of action, and lack of pulmonary selectivity result in noncompliance and poor patient outcomes. In this study, we tested the hypothesis that encapsulation of an investigational anti-PAH molecule fasudil (HA-1077), a Rho-kinase inhibitor, into liposomal vesicles results in prolonged vasodilation in distal pulmonary arterioles. Liposomes were prepared by hydration and extrusion method and fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient. Liposomes were then characterized for various physicochemical properties. Optimized formulations were tested for pulmonary absorption and their pharmacological efficacy in a monocrotaline (MCT) induced rat model of PAH. The entrapment efficiency of optimized liposomal fasudil formulations was between 68.1±0.8% and 73.6±2.3%, and the cumulative release at 37°C was 98–99% over a period of 5 days. Compared to intravenous (IV) fasudil, a ~10 fold increase in the terminal plasma half-life was observed when liposomal fasudil was administered as aerosols. The t1/2 of IV fasudil was 0.39±0.12 h. and when given as liposomes via pulmonary route, the t1/2 extended to 4.71±0.72 h. One h after intratracheal instillation of liposomal fasudil, mean pulmonary arterial pressure (MPAP) was reduced by 37.6±5.7% and continued to decrease for about 3 h, suggesting that liposomal formulations produced pulmonary preferential vasodilation in MCT induced PAH rats. Overall, this study established the proof-of-principle that aerosolized liposomal fasudil is a feasible option for a non-invasive, controlled release and pulmonary preferential treatment of PAH.
PMCID: PMC3632285  PMID: 23353807
Fasudil; liposomes; ammonium sulfate; pulmonary delivery; pulmonary arterial pressure
18.  Manganese-loaded lipid-micellar theranostics for simultaneous drug and gene delivery to lungs 
Gadolinium (Gd) contrast agents are predominantly used for T1 MR imaging. However, the high toxicity of Gd3+ and potential side effects including nephrogenic systemic fibrosis have led to the search for alternative T1 contrast agents. Since manganese (Mn) has paramagnetic properties with five unpaired electrons that permit high spin number, long electronic relaxation times, and labile water exchange, we evaluated Mn as a T1 magnetic resonance imaging (MRI) contrast agent for lung imaging. Here we report on the design and synthesis of multifunctional lipid-micellar nanoparticles (LMNs) containing Mn oxide (M-LMNs) for MRI that can also be used for DNA and drug delivery. Oleic acid-coated MnO nanoparticles were encapsulated in micelles composed of polyethylene glycol (PEG-2000), phosphatidylethanolamine (PE), DC-cholesterol, and dioleoyl-phosphatidylethanolamine (DOPE). The particles are taken up in vitro by human embryonic kidney (HEK293), Lewis lung carcinoma (LLC1), and A549 cells and are devoid of cytotoxicity. When administered to mice intranasally, they preferentially accumulate in the lungs. In vitro phantom and ex vivo lung MRI results confirmed that M-LMNs are able to enhance T1 MRI contrast. M-LMNs loaded with plasmid DNA and/or doxorubicin are efficiently taken up by HEK293 cells in vitro and by target cells in vivo. Taken together, these results demonstrate that M-LMNs are capable of simultaneously providing MRI contrast and DNA and/or drug delivery to target cells in the lung and therefore may prove useful as a lung theranostic, especially for lung cancers.
PMCID: PMC3783961  PMID: 23395689
Theranostics; Micelle; Manganese; Drug; Gene; Delivery
19.  Nanoparticle diffusion in, and microrheology of, the bovine vitreous ex vivo 
Intravitreal injection of biodegradable nanoparticles (NP) holds promise for gene therapy and drug delivery to the back of the eye. In some cases, including gene therapy, NP need to diffuse rapidly from the site of injection in order to reach targeted cell types in the back of the eye, whereas in other cases it may be preferred for the particles to remain at the injection site and slowly release drugs that may then diffuse to the site of action. We studied the movements of polystyrene (PS) nanoparticles of various sizes and surface chemistries in fresh bovine vitreous. PS NP as large as 510 nm rapidly penetrated the vitreous gel when coated with polyethylene glycol (PEG), whereas the movements of NP 1190 nm in diameter or larger were highly restricted regardless of surface chemistry owing to steric obstruction. PS NP coated with primary amine groups (–NH2) possessed positively charged surfaces at the pH of bovine vitreous (pH = 7.2), and were immobilized within the vitreous gel. In comparison, PS NP coated with –COOH (possessing negatively charged surfaces) in the size range of 100–200 nm and at particle concentrations below 0.0025% (w/v) readily diffused through the vitreous meshwork; at higher concentrations (~0.1% w/v), these nanoparticles aggregated within vitreous. Based on the mobility of different sized PS-PEG NP, we estimated the average mesh size of fresh bovine vitreous to be ~550 ± 50 nm. The bovine vitreous behaved as an impermeable elastic barrier to objects sized 1190 nm and larger, but as a highly permeable viscoelastic liquid to non-adhesive objects smaller than 510 nm in diameter. Guided by these studies, we next sought to examine the transport of drug- and DNA-loaded nanoparticles in bovine vitreous. Biodegradable NP with diameter of 227 nm, composed of a poly(lactic-co-glycolic acid) (PLGA)-based core coated with poly(vinyl alcohol) rapidly penetrated vitreous. Rod-shaped, highly-compacted CK30PEG10k/DNA with PEG coating (neutral surface charge; diameter ~60 nm) diffused rapidly within vitreous. These findings will help guide the development of nanoparticle-based therapeutics for the treatment of vision-threatening ocular diseases.
PMCID: PMC3693951  PMID: 23369761
Eye; Nanotechnology; Drug delivery; Gene delivery; Particle tracking
20.  Crosslinked Linear Polyethyleneimine Enhances Delivery of DNA to the Cytoplasm 
Crosslinked polyethylenimines (PEIs) have been frequently examined over the past decade since they can maintain the transfection efficiency of commercially available, 25k branched PEI, but exhibit less cytotoxicity. The argument is often made that the degradability of such polymers, generally synthesized with either disulfide or hydrolytically degradable crosslinkers, is critical to the high efficiency and low toxicity of the system. In this work, we present a crosslinked linear PEI (xLPEI) system in which either disulfide-responsive or non-degradable linkages are incorporated. As with previous systems, strong transfection efficiency in comparison with commercial standards was achieved with low cytotoxicity. However, these properties were shown to be present when either the degradable or non-degradable crosslinker was used. Uncomplexed polymer was demonstrated to be the critical factor determining transfection efficiency for these polymers, mediating efficient endosomal escape without signs of cell membrane damage. While several crosslinked PEI systems in the literature have demonstrated the effect of the disulfide moiety, this work demonstrates that disulfide-mediated unpackaging may not be as important as conventionally thought for some PEI systems.
PMCID: PMC3914540  PMID: 22995755
Gene Delivery; Polyethylenimine; Bioresponsive; Reducible; Endosomal Escape
21.  Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes 
Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 μg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with 125I and the local retention of 125I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (μCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of 125I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo μCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.
PMCID: PMC3974410  PMID: 19105972
Drug delivery; Controlled release; Bone morphogenetic protein-2; Single photon emission computed; tomography; Scintillation probes; Micro-computed tomography
22.  Polymalic acid nanobioconjugate for simultaneous inhibition of tumor growth and immunostimulation in HER2/neu-positive breast cancer✩ 
Breast cancer remains the second leading cause of cancer death among women in the United States. The breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensuring the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retaining the biological activity of IL-2. We also showed the uptake of the nanobioconjugate by HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. In addition, the nanobioconjugate was capable of eliciting anti-tumor activity in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. Both IgG1 and IgG2a levels were significantly increased in animals treated with the PMLA-fusion nanobioconjugate compared to animals treated with the antibody–cytokine fusion protein alone or control animals, indicative of the induction of a humoral (TH2) and cell-mediated (TH1) immune responses. Animal survival in vivo was significantly longer after treatment with leading nanobioconjugate with fusion [anti-HER2/neu IgG3-(IL-2)] antibody, p < 0.05. The combination of these molecules on a single polymeric platform is expected to act through direct elimination of cancer cells, inhibition of tumor angiogenesis, and orchestration of a potent immune response against tumor.
PMCID: PMC3971991  PMID: 23770212
Polymalic acid; Nanobioconjugate; Nanopolymer; HER2/neu; Antibody fusion protein; IL-2; Laminin-411; Breast cancer
23.  Transfection Efficiency and Transgene Expression Kinetics of mRNA Delivered in Naked and Nanoparticle Format 
Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. Protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7 hours and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4 hours and lasts less than 24 hours, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18 hours and persists for at least 6 days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.
PMCID: PMC3594075  PMID: 23306021
mRNA; nanoparticle; kinetics; non-viral gene therapy; transfection; dendritic cell
24.  Gene silencing following siRNA delivery to skin via coated steel microneedles: in vitro and in vivo proof-of-concept 
The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified “self-delivery” siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin.
PMCID: PMC3594125  PMID: 23313112
Microneedles; Coating; Lipoplex; Self-delivery siRNA; Keratinocytes; RNA interference
25.  Stability of whole inactivated influenza virus vaccine during coating onto metal microneedles 
Immunization using a microneedle patch coated with vaccine offers the promise of simplified vaccination logistics and increased vaccine immunogenicity. This study examined the stability of influenza vaccine during the microneedle coating process, with a focus on the role of coating formulation excipients. Thick, uniform coatings were obtained using coating formulations containing a viscosity enhancer and surfactant, but these formulations retained little functional vaccine hemagglutinin (HA) activity after coating. Vaccine coating in a trehalose-only formulation retained about 40 – 50% of vaccine activity, which is a significant improvement. The partial viral activity loss observed in the trehalose-only formulation was hypothesized to come from osmotic pressure-induced vaccine destabilization. We found that inclusion of a viscosity enhancer, carboxymethyl cellulose, overcame this effect and retained full vaccine activity on both washed and plasma-cleaned titanium surfaces. The addition of polymeric surfactant, Lutrol® micro 68, to the trehalose formulation generated phase transformations of the vaccine coating, such as crystallization and phase separation, which was correlated to additional vaccine activity loss, especially when coating on hydrophilic, plasma-cleaned titanium. Again, the addition of a viscosity enhancer suppressed the surfactant-induced phase transformations during drying, which was confirmed by in vivo assessment of antibody response and survival rate after immunization in mice. We conclude that trehalose and a viscosity enhancer are beneficial coating excipients, but the inclusion of surfactant is detrimental to vaccine stability.
PMCID: PMC3578180  PMID: 23246470
Coated microneedle patch; Skin vaccination; Influenza vaccine stability; Coating formulation; Crystallization; Phase separation; Osmotic pressure

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