Bioinspired Nanoparticles Engineered for Enhanced Delivery to the Bone.

Targeting therapeutic agents to specific organs in the body remains a challenge despite advances in the science of systemic drug delivery. We have engineered a programmable-bioinspired nanoparticle (P-BiNP) delivery system to simultaneously target the bone and increase uptake in homotypic tumor cells by coating polymeric nanoparticles with programmed cancer cell membranes. This approach is unique in that we have incorporated relevant clinical bioinformatics data to guide the design and enhancement of biological processes that these nanoparticles are engineered to mimic. To achieve this, an analysis of RNA expression from metastatic prostate cancer patients identified ITGB3 (a subunit of integrin α V ß 3) as overexpressed in patients with bone metastasis. Cancer cells were stimulated to increase this integrin expression on the cell surface, and these membranes were subsequently used to coat cargo carrying polymeric nanoparticles. Physicochemical optimization and characterization of the P-BiNPs showed desirable qualities regarding size, ζ potential, and stability. In vitro testing confirmed enhanced homotypic binding and uptake in cancer cells. P-BiNPs also demonstrated improved bone localization in vivo with a murine model. This novel approach of identifying clinically relevant targets for dual homotypic and bone targeting has potential as a strategy for treatment and imaging modalities in diseases that affect the bone as well as broader implications for delivering nanoparticles to other organs of interest.

"Curcumin-loaded Poly (d, l-lactide-co-glycolide) nanovesicles induce antinociceptive effects and reduce pronociceptive cytokine and BDNF release in spinal cord after acute administration in mice".

Given the poor bioavailability of curcumin, its antinociceptive effects are produced after chronic intravenous administration of high doses, while poly (d,l-lactide-co-glycolide)-loaded vesicles (PLGA) can improve drug delivery. This paper investigates the antinociceptive effects of curcumin-loaded PLGA nanovesicles (PLGA-CUR) administered via intravenous (i.v.) or intrathecal (i.t.) routes at low and high doses. The following models of pain were used: formalin test, zymosan-induced hyperalgesia and sciatic nerve ligation inducing neuropathic allodynia and hyperalgesia. PLGA-CUR administered intravenously was able to reduce the response to nociceptive stimuli in the formalin test and hyperalgesia induced by zymosan. Curcumin, instead, was inactive. Low-dose i.t. administration of PLGA-CUR significantly reduced allodynia produced by sciatic nerve ligation, whereas low doses of curcumin did not change the response to nociceptive stimuli. Long-lasting antinociceptive effects were observed when high doses of PLGA-CUR were administered intrathecally. At high doses, i.t. administration of curcumin only exerted rapid and transient antinociceptive effects. Measurement of cytokine and BDNF in the spinal cord of neuropathic mice demonstrate that the antinociceptive effects of PLGA-CUR depend on the reduction in cytokine release and BDNF in the spinal cord. The results demonstrate the effectiveness of PLGA-CUR and suggest that PLGA-CUR nanoformulation might be a new potential drug in the treatment of pain.

Curcumin-ER Prolonged Subcutaneous Delivery for the Treatment of Non-Small Cell Lung Cancer.

Non-small-cell lung cancer therapy is a challenge due to poor prognosis and low survival rate. There is an acute need for advanced therapies having higher drug efficacy, low immunogenicity and fewer side effects which will markedly improve patient compliance and quality of life of cancer patients. The purpose of this study was to develop a novel hybrid curcumin nanoformulation (Curcumin-ER) and evaluate the therapeutic efficacy of this formulation on a non-small cell lung cancer xenograft model. Use of curcumin, a natural anticancer agent, is majorly limited due to its poor aqueous solubility and hence it's low systemic bioavailability. In this paper, we carried out the nanoformulation of Curcumin-ER, optimized the formulation process and determined the anticancer effects of Curcumin-ER against human A549 non-small cell lung cancer using in vitro and in vivo studies. Xenograft tumors in nude mice were treated with 20 mg/kg subcutaneous injection of Curcumin-ER and liposomal curcumin (Lipocurc) twice a week for seven weeks. Results showed that tumor growth was suppressed by 52.1% by Curcumin-ER treatment and only 32.2% by Lipocurc compared to controls. Tumor sections were isolated from murine xenografts and histology and immunohistochemistry was performed. A decrease in expression of NFκB-p65 subunit and proliferation marker, Ki-67 was observed in treated tumors. In addition, a potent anti-angiogenic effect, characterized by reduced expression of annexin A2 protein, was observed in treated tumors. These results establish the effectiveness of Curcumin-ER in regressing human non-small cell lung cancer growth in the xenograft model using subcutaneous route of administration. The therapeutic efficacy of Curcumin-ER highlights the potential of this hybrid nanoformulation in treating patients with non-small cell lung cancer.

Targeted Nanocurcumin Therapy Using Annexin A2 Anitbody Improves Tumor Accumulation and Therapeutic Efficacy Against Highly Metastatic Breast Cancer.

A major challenge in pharmaceutical research is effective targeting strategies to their sites of action. Emerging knowledge and the current progress in nanotechnology based delivery systems has opened up exciting ways towards successful targeted nanodelivery systems. For cancer therapy, nanoparticle-based drug formulations hold several advantages over free drugs, including improved pharmacokinetics, enhanced tumor accumulation, reduced systemic exposure and side effects and better patient compliance. The goal of this study was to validate the in vivo targeting potential and evaluate the combinatorial therapeutic potential of novel Annexin A2 (AnxA2) antibody-conjugated curcumin loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (AnxA2-CPNP) against metastatic breast cancer. As a first step, we demonstrated that the cell-surface expression of AnxA2 is increases during breast cancer progression with very high expression in highly malignant cancer cells and basal expression in non-malignant cells. This confirmed AnxA2 as an excellent target for targeting our curcumin nanoparticles. Our results indicate that AnxA2-CPNP showed increased uptake in highly metastatic breast cancer cells than untargeted nanoparticles due to the differential AnxA2 expression. Cell viability, plasmin generation and wound healing assays reveal that AnxA2-CPNPs effectively inhibited cell proliferation, invasion and migration, key elements for cancer growth and metastasis. Further, angiogenesis assay illustrated that AnxA2-CPNPs decreased the formation of tube capillaries, thus inhibiting neoangiogenesis, a critical element in tumor growth. Live animal imaging demonstrated that AnxA2-PNPs and AnxA2-CPNPs effectively targeted and accumulated in the tumor as seen by the increased fluorescence intensity on the live scans. Xenograft studies in mice showed significant regression of breast tumor as a result of both effective targeting, accumulation and sustained release of curcumin in the tumor. In conclusion, AnxA2-CPNPs were successfully validated for their breast tumor targeting potential and its improved therapeutic efficacy against metastatic breast cancer.

A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having µM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

Development of biodegradable nanocarriers loaded with a monoclonal antibody.

Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%-22% and antibody loading of 0.3%-1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells.

Nanobiosensors: role in cancer detection and diagnosis.

The ability to detect many cancers at an early stage in its clinical course has the potential to improve patient outcomes in terms of morbidity and mortality. Nanosized components incorporated into existing clinical diagnostic and detection systems as well as novel nanobiosensors have demonstrated improved sensitivity and specificity compared with traditional cancer testing approaches. Nanoparticles, nanowires, nanotubes, and nanocantilevers are examples of four nanobiosensor systems that have been used experimentally in the context of detection and diagnosis of prostate, breast, pancreatic, lung, and brain cancers over the past few years. Nanobiosensors will begin to transition into clinically validated tests as experimental and engineering techniques advance. This paper presents examples of some such nanobiosensors for cancer diagnosis and detection.

Fluorescence detection of MMP-9. II. Ratiometric FRET-based sensing with dually labeled specific peptide.

In our previous paper we showed that the MMP-9 enzyme recognizes a specific peptide sequence, Lys-Gly- Pro-Arg-Ser-Leu-Ser-Gly-Lys, and cleaves the peptide into two parts [1]. In this study, the peptide is labeled with two dyes, carboxyfluorescein (5-FAM) and Cy5. A highly efficient energy transfer of over 80% results in a dominant emission of Cy5 at ~670 nm with an excitation of 470 nm. Severance of the peptide by the MMP-9 enzyme eliminates Förster Resonance Energy Transfer (FRET) and strongly increases the fluorescence of the 5-FAM dye. In this manuscript we describe the strategy for a FRET-based method for MMP-9 enzyme detection. The basic aim is to apply a ratio-metric sensing technique in which a ratio of green/red fluorescence intensity is measured as a function of enzyme concentration. The ratio-metric method eliminates many experimental variables and enables accurate MMP-9 detection.

Mitigating prolonged QT interval in cancer nanodrug development for accelerated clinical translation.

BACKGROUND: Cardiac toxicity is the foremost reason for drug discontinuation from development to clinical evaluation and post market surveillance [Fung 35:293-317, 2001; Piccini 158:317-326 2009]. The Food and Drug Administration (FDA) has rejected many potential pharmaceutical agents due to QT prolongation effects. Since drug development and FDA approval takes an enormous amount of time, money and effort with high failure rates, there is an increased focus on rescuing drugs that cause QT prolongation. If these otherwise safe and potent drugs were formulated in a unique way so as to mitigate the QT prolongation associated with them, these potent drugs may get FDA approval for clinical use. Rescuing these compounds not only benefit the patients who need them but also require much less time and money thus leading to faster clinical translation. In this study, we chose curcumin as our drug of choice since it has been shown to posses anti-tumor properties against various cancers with limited toxicity. The major limitations with this pharmacologically active drug are (a) its ability to prolong QT by inhibiting the hERG channel and (b) its low bioavailability. In our previous studies, we found that lipids have protective actions against hERG channel inhibition and therefore QT prolongation. RESULTS: Results of the manual patch clamp assay of HEK 293 cells clearly illustrated that our hybrid nanocurcumin formulation prevented the curcumin induced inhibition of hERG K+ channel at concentrations higher than the therapeutic concentrations of curcumin. Comparing the percent inhibition, the hybrid nanocurcumin limited inhibition to 24.8% at a high curcumin equivalent concentration of 18 µM. Liposomal curcumin could only decrease this inhibition upto 30% only at lower curcumin concentration of 6 µM but not at 18 µM concentration. CONCLUSIONS: Here we show a curcumin encapsulated lipopolymeric hybrid nanoparticle formulation which could protect against QT prolongation and also render increased bioavailability and stability thereby overcoming the limitations associated with curcumin.

Efficacy of liposomal curcumin in a human pancreatic tumor xenograft model: inhibition of tumor growth and angiogenesis.

BACKGROUND: Liposome-based drug delivery has been successful in the past decade, with some formulations being Food and Drug Administration (FDA)-approved and others in clinical trials around the world. The major disadvantage associated with curcumin, a potent anticancer agent, is its poor aqueous solubility and hence low systemic bioavailability. However, curcumin can be encapsulated into liposomes to improve systemic bioavailability. MATERIALS AND METHODS: We determined the antitumor effects of a liposomal curcumin formulation against human MiaPaCa pancreatic cancer cells both in vitro and in xenograft studies. Histological sections were isolated from murine xenografts and immunohistochemistry was performed. RESULTS: The in vitro (IC50) liposomal curcumin proliferation-inhibiting concentration was 17.5 µM. In xenograft tumors in nude mice, liposomal curcumin at 20 mg/kg i.p. three-times a week for four weeks induced 42% suppression of tumor growth compared to untreated controls. A potent antiangiogenic effect characterized by a reduced number of blood vessels and reduced expression of vascular endothelial growth factor and annexin A2 proteins, as determined by immunohistochemistry was observed in treated tumors. CONCLUSION: These data clearly establish the efficacy of liposomal curcumin in reducing human pancreatic cancer growth in the examined model. The therapeutic curcumin-based effects, with no limiting side-effects, suggest that liposomal curcumin may be beneficial in patients with pancreatic cancer.

Scale up, optimization and stability analysis of Curcumin C3 complex-loaded nanoparticles for cancer therapy.

BACKGROUND: Nanoparticle based delivery of anticancer drugs have been widely investigated. However, a very important process for Research & Development in any pharmaceutical industry is scaling nanoparticle formulation techniques so as to produce large batches for preclinical and clinical trials. This process is not only critical but also difficult as it involves various formulation parameters to be modulated all in the same process. METHODS: In our present study, we formulated curcumin loaded poly (lactic acid-co-glycolic acid) nanoparticles (PLGA-CURC). This improved the bioavailability of curcumin, a potent natural anticancer drug, making it suitable for cancer therapy. Post formulation, we optimized our process by Reponse Surface Methodology (RSM) using Central Composite Design (CCD) and scaled up the formulation process in four stages with final scale-up process yielding 5 g of curcumin loaded nanoparticles within the laboratory setup. The nanoparticles formed after scale-up process were characterized for particle size, drug loading and encapsulation efficiency, surface morphology, in vitro release kinetics and pharmacokinetics. Stability analysis and gamma sterilization were also carried out. RESULTS: Results revealed that that process scale-up is being mastered for elaboration to 5 g level. The mean nanoparticle size of the scaled up batch was found to be 158.5±9.8 nm and the drug loading was determined to be 10.32±1.4%. The in vitro release study illustrated a slow sustained release corresponding to 75% drug over a period of 10 days. The pharmacokinetic profile of PLGA-CURC in rats following i.v. administration showed two compartmental model with the area under the curve (AUC0-∞) being 6.139 mg/L h. Gamma sterilization showed no significant change in the particle size or drug loading of the nanoparticles. Stability analysis revealed long term physiochemical stability of the PLGA-CURC formulation. CONCLUSIONS: A successful effort towards formulating, optimizing and scaling up PLGA-CURC by using Solid-Oil/Water emulsion technique was demonstrated. The process used CCD-RSM for optimization and further scaled up to produce 5 g of PLGA-CURC with almost similar physicochemical characteristics as that of the primary formulated batch.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells.

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

A sustained release formulation of chitosan modified PLCL:poloxamer blend nanoparticles loaded with optical agent for animal imaging.

The objective of this study was to develop optical imaging agent loaded biodegradable nanoparticles with indocynanine green (ICG) using chitosan modified poly(L-lactide-co-epsilon-caprolactone) (PLCL):poloxamer (Pluronic F68) blended polymer. Nanoparticles were formulated with an emulsification solvent diffusion technique using PLCL and poloxamer as blend-polymers. Polyvinyl alcohol (PVA) and chitosan were used as stabilizers. The particle size, shape and zeta potential of the formulated nanoparticles and the release kinetics of ICG from these nanoparticles were determined. Further, biodistribution of these nanoparticles was studied in mice at various time points until 24 h following intravenous administration, using a non-invasive imaging system. The average particle size of the nanoparticles was found to be 146 ± 3.7 to 260 ± 4.5 nm. The zeta potential progressively increased from - 41.6 to + 25.3 mV with increasing amounts of chitosan. Particle size and shape of the nanoparticles were studied using transmission electron microscopy (TEM) which revealed the particles to be smooth and spherical in shape. These nanoparticles were efficiently delivered to the cytoplasm of the cells, as observed in prostate and breast cancer cells using confocal laser scanning microscopy. In vitro release studies indicated sustained release of ICG from the nanoparticles over a period of seven days. Nanoparticle distribution results in mice showing improved uptake and accumulation with chitosan modified nanoparticles in various organs and slower clearance at different time points over a 24 h period as compared to unmodified nanoparticles. The successful formulation of such cationically modified nanoparticles for encapsulating optical agents may lead to a potential deep tissue imaging technique for tumor detection, diagnosis and therapy.

Fluorescence detection of MMP-9. I. MMP-9 selectively cleaves Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys peptide.

MMP-9 enzyme recognizes a peptide sequence Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys and cleaves the peptide into two parts. We synthesized a dual fluorophore beacon consisting of 5-FAM and Cy5 dyes. The fluorescence emission of the fluorescein moiety is dramatically quenched by Cy5 molecule due to Förster Resonance Energy Transfer (FRET) and the fluorescence of Cy5 is strongly enhanced. Upon addition of MMP-9 enzyme, the fluorescence of 5-FAM intensifies and Cy5 decreases. The control MMP-2 enzyme does not cause any changes in either 5-FAM or Cy5 fluorescence. We believe that our observation will help in early detection of elevated MMP-9 levels under disease conditions.

Spectroscopic properties of curcumin: orientation of transition moments.

Curcumin, a naturally occurring yellow-orange pigment with potent antioxidant and antitumor properties, has been attracting researchers from a wide range of fields including chemistry, spectroscopy, biology, and medicine. Ultrafast excited-state processes such as solvation and excited-state intramolecular hydrogen atom transfer (ESIHT) make curcumin an attractive agent for photodynamic therapy. In this report we present studies of linear dichroism and fluorescence anisotropy in oriented and isotropic media. The results show transition moments (long wavelength absorption and emission) oriented along the long molecular axis. Comparison of linear dichroism and excitation anisotropy in oriented and isotropic media suggests that excited-state intramolecular hydrogen atom transfer is probably associated with intramolecular conformational changes that can be constrained in highly stretched poly(vinyl alcohol) (PVA) film.

Enhanced fluorescence of curcumin on plasmonic platforms.

We report a significant increase of a curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione) fluorescence brightness when deposited on plasmonic platforms (self assembled silver nanostructures formed on the surface of silver semitransparent film). The enhancement of fluorescence intensity is accompanied by a strong decrease in fluorescence lifetime. Simultaneously, the increased photostability of curcumin, a pigment showing strong anti-inflammatory, antioxidant and antitumorigenic activity, allows long-time detection and monitoring. We believe that the use of plasmonic platforms will improve detection, delivery and imaging of curcumin.

Formulation, characterization and evaluation of curcumin-loaded PLGA nanospheres for cancer therapy.

BACKGROUND: Among the potent anticancer agents, curcumin has been found to be very efficacious against many different types of cancer cells. However, the major disadvantage associated with the use of curcumin is its low systemic bioavailability when administered orally due to its poor aqueous solubility. Our present work investigated the efficiency of encapsulation of curcumin in poly (lactic-coglycolic acid) (PLGA) nanospheres using solid/oil/water emulsion solvent evaporation method. MATERIALS AND METHODS: The nanospheres were formulated and then characterized for percent yield, encapsulation efficiency, surface morphology, particle size, drug distribution studies, drug polymer interaction studies and in vitro drug release profiles. RESULTS: Our studies showed the successful formation of smooth and spherical curcumin-loaded PLGA nanospheres, with an encapsulation efficiency of 90.88+/-0.14%. The particle size distribution showed a range of 35 nm to 100 nm, with the mean particle size being 45 nm. Evaluation of these curcumin-loaded nanospheres was carried out in prostate cancer cell lines. Results showed robust intracellular uptake of the nanospheres in the cells. Cell viability studies revealed that the curcumin-loaded nanospheres were able to exert a more pronounced effect on the cancer cells as compared to free curcumin. CONCLUSION: Our studies achieved successful formulation of curcumin loaded PLGA nanospheres, thus indicating that nanoparticle-based formulation of curcumin has high potential as an adjuvant therapy for clinical application in prostate cancer.