Leveraging Surface Plasmon Resonance to Dissect the Interfacial Properties of Nanoparticles: Implications for Tissue Binding and Tumor Penetration.

Therapeutic efficacy of nanoparticle-drug formulations for cancer applications is significantly impacted by the extent of intra-tumoral accumulation and tumor tissue penetration. We advanced the application of surface plasmon resonance to examine interfacial properties of various clinical and emerging nanoparticles related to tumor tissue penetration. We observed that amine-terminated or positively-charged dendrimers and liposomes bound strongly to tumor extracellular matrix (ECM) proteins, whereas hydroxyl/carboxyl-terminated dendrimers and PEGylated/neutrally-charged liposomes did not bind. In addition, poly(lactic-co-glycolic acid) (PLGA) nanoparticles formulated with cholic acid or F127 surfactants bound strongly to tumor ECM proteins, whereas nanoparticles formulated with poly(vinyl alcohol) did not bind. Unexpectedly, following blood serum incubation, this binding increased and particle transport in ex vivo tumor tissues reduced markedly. Finally, we characterized the protein corona on PLGA nanoparticles using quantitative proteomics. Through these studies, we identified valuable criteria for particle surface characteristics that are likely to mediate their tissue binding and tumor penetration.

Sustained release of calcium hydroxide from poly(DL-lactide-co-glycolide) acid microspheres for apexification.

Calcium hydroxide (CH) loaded poly(DL-lactide-co-glycolide) acid (PLGA) microspheres (MS) might be used for apexification requiring a sustained release of Ca(2+). The aim of this study was to formulate and characterize CH-PLGA-MS. The CH-loaded MS were prepared by either oil-in-water (O/W) or water-in-oil/in-water (W/O/W) emulsion solvent evaporation technique. MS produced by the O/W technique exhibited a larger diameter (18.63 ± 7.23 µm) than the MS produced by the W/O/W technique (15.25 ± 7.37 µm) (Mann-Whitney U test P < 0.001). The CH encapsulation efficiency (E e) and Ca(2+) release were calculated from data obtained by absorption techniques. Ca(2+) release profile was evaluated for 30 days. To know the E e, the CH-loaded MS were dissolved in 1 M NaOH to release all its content and a Ca(2+) colorimetric marker was added to this solution. The reagent marked the Ca(2+) in blue color, which was then measured by a UV-Vis system (650 nm). The percentage of E e was calculated on the basis of the theoretical loading. The E e of the O/W-produced MS was higher (24 %) than the corresponding percentage of the W/O/W-produced MS (11 %). O/W- and W/O/W-produced MS released slower and lower Ca(2+) than a control CH paste with polyethylene glycol 400 (Kruskal-Wallis test). O/W-produced MS released higher Ca(2+) than W/O/W-produced MS (statistically significant differences; P < 0.05). In conclusion, the CH-PLGA-MS were successfully formulated; the technique of formulation influenced the size, encapsulation efficiency and release profile. The MS were better sustained release system than the CH paste.

Synthesis and characterization of a novel pH-responsive drug-releasing nanocomposite hydrogel for skin cancer therapy and wound healing.

Local skin cancer recurrence occurs in ∼12% of the patients post-surgery due to persistent growth of residual cancer cells. Wound infection is another significant complication following surgery. We report a novel in situ-forming nanocomposite hydrogel (NCH) containing PLGA-carboxymethyl chitosan nanoparticles (186 nm) for localized pH-responsive skin cancer therapy and wound healing. This injectable hydrogel, comprising of a citric acid-derived polymer backbone, gelled within 5 minutes, and demonstrated excellent swelling (283% of dry weight) and compressive strengths (∼5.34 MPa). Nanoparticle incorporation did not significantly affect hydrogel properties. The NCH effluents were cytocompatible with human dermal fibroblasts at 500 µg ml-1 concentration and demonstrated pH-dependent drug release and promising therapeutic efficacy against A431 and G361 skin cancer cells in vitro. Significant zones of inhibition were observed in S. aureus and E. coli cultures on NCH treatment, confirming its antibacterial properties. Our studies show that the pH-responsive NCH can be potentially used for adjuvant skin cancer treatment and wound healing.

Factorial analyses of photopolymerizable thermoresponsive composite hydrogels for protein delivery.

A smart protein delivery system for wound healing applications was developed using composite nanoparticle hydrogels that can release protein in a temperature-responsive manner. This system can also be formed in situ in the presence of ultraviolet light and Irgacure 2959 photoinitiator. The system consists of temperature-sensitive poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAm) nanoparticles embedded in a poly(ethylene glycol) diacrylate (PEGDA) matrix. A factorial analysis was performed to evaluate the effects of PEGDA concentration (10% and 15% w/v) and PEGDA molecular weight (MW; 3.4 kDa and 8 kDa), as well as PNIPAM-AAm nanoparticle concentration (2% and 4% w/v) and temperature (23 degrees C and 40 degrees C) on the protein release profiles and swelling ratios of the hydrogels. Results indicate that PNIPAM-AAm nanoparticle concentration and temperature were the most important factors affecting the protein release during the burst release phase. Additionally, PEGDA MW was the most important factor affecting the protein release in the plateau region. It was also important in controlling the hydrogel swelling ratio. A dual-layered hydrogel was further developed to produce a protein delivery system with a better sustained release. These findings have improved our understanding of the composite hydrogel systems and will help in tailoring future systems with desired release profiles. FROM THE CLINICAL EDITOR: A smart protein delivery system for wound healing applications using composite nanoparticle hydrogels that can release protein in a temperature-responsive manner is reported in this paper. Systems like this may aid in optimal would healing in the surgical and trauma-related settings.

Decreased non-specific adhesivity, receptor targeted (DART) nanoparticles exhibit improved dispersion, cellular uptake, and tumor retention in invasive gliomas.

The most common and deadly form of primary brain cancer, glioblastoma (GBM), is characterized by significant intratumoral heterogeneity, microvascular proliferation, immune system suppression, and brain tissue invasion. Delivering effective and sustained treatments to the invasive GBM cells intermixed with functioning neural elements is a major goal of advanced therapeutic systems for brain cancer. Previously, we investigated the nanoparticle characteristics that enable targeting of invasive GBM cells. This revealed the importance of minimizing non-specific binding within the relatively adhesive, 'sticky' microenvironment of the brain and brain tumors in particular. We refer to such nanoformulations with decreased non-specific adhesivity and receptor targeting as 'DART' therapeutics. In this work, we applied this information toward the design and characterization of biodegradable nanocarriers, and in vivo testing in orthotopic experimental gliomas. We formulated particulate nanocarriers using poly(lactic-co-glycolic acid) (PLGA) and PLGA-polyethylene glycol (PLGA-PEG) polymers to generate sub-100nm nanoparticles with minimal binding to extracellular brain components and strong binding to the Fn14 receptor - an upregulated, conserved component in invasive GBM. Multiple particle tracking in brain tissue slices and in vivo testing in orthotopic murine malignant glioma revealed preserved nanoparticle diffusivity and increased uptake in brain tumor cells. These combined characteristics also resulted in longer retention of the DART nanoparticles within the orthotopic tumors compared to non-targeted versions. Taken together, these results and nanoparticle design considerations offer promising new methods to optimize therapeutic nanocarriers for improving drug delivery and treatment for invasive brain tumors.

Non-specific binding and steric hindrance thresholds for penetration of particulate drug carriers within tumor tissue.

Therapeutic nanoparticles (NPs) approved for clinical use in solid tumor therapy provide only modest improvements in patient survival, in part due to physiological barriers that limit delivery of the particles throughout the entire tumor. Here, we explore the thresholds for NP size and surface poly(ethylene glycol) (PEG) density for penetration within tumor tissue extracellular matrix (ECM). We found that NPs as large as 62nm, but less than 110nm in diameter, diffused rapidly within a tumor ECM preparation (Matrigel) and breast tumor xenograft slices ex vivo. Studies of PEG-density revealed that increasing PEG density enhanced NP diffusion and that PEG density below a critical value led to adhesion of NP to ECM. Non-specific binding of NPs to tumor ECM components was assessed by surface plasmon resonance (SPR), which revealed excellent correlation with the particle diffusion results. Intravital microscopy of NP spread in breast tumor tissue confirmed a significant difference in tumor tissue penetration between the 62 and 110nm PEG-coated NPs, as well as between PEG-coated and uncoated NPs. SPR assays also revealed that Abraxane, an FDA-approved non-PEGylated NP formulation used for cancer therapy, binds to tumor ECM. Our results establish limitations on the size and surface PEG density parameters required to achieve uniform and broad dispersion within tumor tissue and highlight the utility of SPR as a high throughput method to screen NPs for tumor penetration.

Design of antimicrobial peptides conjugated biodegradable citric acid derived hydrogels for wound healing.

Wound healing is usually facilitated by the use of a wound dressing that can be easily applied to cover the wound bed, maintain moisture, and avoid bacterial infection. In order to meet all of these requirements, we developed an in situ forming biodegradable hydrogel (iFBH) system composed of a newly developed combination of biodegradable poly(ethylene glycol) maleate citrate (PEGMC) and poly(ethylene glycol) diacrylate (PEGDA). The in situ forming hydrogel systems are able to conform to the wound shape in order to cover the wound completely and prevent bacterial invasion. A 2(k) factorial analysis was performed to examine the effects of polymer composition on specific properties, including the curing time, Young's modulus, swelling ratio, and degradation rate. An optimized iFBH formulation was achieved from the systematic factorial analysis. Further, in vitro biocompatibility studies using adult human dermal fibroblasts (HDFs) confirmed that the hydrogels and degradation products are not cytotoxic. The iFBH wound dressing was conjugated and functionalized with antimicrobial peptides as well. Evaluation against bacteria both in vitro and in vivo in rats demonstrated that the peptide-incorporated iFBH wound dressing offered excellent bacteria inhibition and promoted wound healing. These studies indicated that our in situ forming antimicrobial biodegradable hydrogel system is a promising candidate for wound treatment.

Dual-responsive polymer-coated iron oxide nanoparticles for drug delivery and imaging applications.

We reported the synthesis and characterization of dual-responsive poly(N-isopropylacrylamide-acrylamide-chitosan) (PAC)-coated magnetic nanoparticles (MNPs) for controlled and targeted drug delivery and imaging applications. The PAC-MNPs size was about 150nm with 70% iron mass content and excellent superparamagnetic properties. PAC-MNPs loaded with anti-cancer drug doxorubicin showed dual-responsive drug release characteristics with the maximum release of drugs at 40°C (∼78%) than at 37°C (∼33%) and at pH of 6 (∼55%) than at pH of 7.4 (∼28%) after 21 days. Further, the conjugation of prostate cancer-specific R11 peptides increased the uptake of PAC-MNPs by prostate cancer PC3 cells. The dose-dependent cellular uptake of the nanoparticles was also significantly increased with the presence of 1.3T magnetic field. The nanoparticles demonstrated cytocompatibility up to concentrations of 500µg/ml when incubated over a period of 24h with human dermal fibroblasts and normal prostate epithelial cells. Finally, pharmacokinetic studies indicated that doxorubicin-loaded PAC-MNPs caused significant prostate cancer cell death at 40°C than at 37°C, thereby confirming the temperature-dependent drug release kinetics and in vitro therapeutic efficacy. Future evaluation of in vivo therapeutic efficacy of targeted image-guided cancer therapy using R11-PAC-MNPs will reinforce a significant impact of the multifunctional PAC-MNPs on the future drug delivery systems.

Prostate cancer-specific thermo-responsive polymer-coated iron oxide nanoparticles.

Thermo-responsive poly(N-isopropylacrylamide-acrylamide-allylamine)-coated magnetic nanoparticles (PMNPs) were developed and conjugated with prostate cancer-specific R11 peptides for active targeting and imaging of prostate cancer. The stable nanoparticles with an average diameter of 100 nm and surface charge of -27.0 mV, had a lower critical solution temperature of 40 °C. Magnetic characterization showed that the nanoparticles can be recruited using a magnetic field and possess superparamagnetic behavior even after R11 conjugation. In vitro cell studies demonstrated that R11-conjugated PMNPs (R11-PMNPs) were compatible with human dermal fibroblasts and normal prostate epithelial cells to all tested concentrations up to 500 µg/ml after 24 h of incubation. Moreover, the nanoparticles were taken up by prostate cancer cells (PC3 and LNCaP) in a dose-dependent manner, which was higher in case of R11-PMNPs than PMNPs. Further, in vivo biodistribution of the nanoparticles showed significantly more R11-PMNPs accumulation in tumors than other vital organs unlike PMNPs without R11 conjugation. Moreover, R11-PMNPs decreased 30% magnetic resonance T2 signal intensity in tumors in vivo compared to 0% decrease with PMNPs. These results indicate great potential of R11-PMPs as platform technology to target and monitor prostate cancers for diagnostic and therapeutic applications.

Effects of surfactants on the properties of PLGA nanoparticles.

The objective of this study was to investigate the physical characteristics of poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) coated with two surfactants, Pluronic or the commonly used polyvinyl alcohol (PVA); and determine their in vitro efficiency as drug carriers for cancer therapy. Free surfactant cytotoxicity results indicated that Pluronic F127 (PF127) was most cytocompatible among the Pluronics tested and hence chosen for coating PLGA NPs for further studies. Release studies using doxorubicin (DOX) as a drug model showed sustained release of DOX from both PVA- and PF127-coated PLGA NPs (PLGA-PVA and PLGA-PF127, respectively) over 28 days. Further, there was no significant difference in human dermal fibroblasts and human aortic smooth muscle cell survival when exposed to both types of NPs. Cellular uptake studies demonstrated that uptake of both nanoparticle types was dose-dependent for both prostate and breast cancer cells. However, these cancer cells internalized more PLGA-PF127 NPs than PLGA-PVA NPs. Moreover, studies showed that drug-loaded PLGA-PF127 NPs not only killed more cancer cells than drug-loaded PLGA-PVA NPs, but also overcame drug resistance in LNCaP, MDA-MB-231, and MDA-MB-468 cancer cells on re-exposure. These results indicate that PLGA-PF127 NPs can form a promising system that not only delivers anti-cancer drugs, but also overcomes drug resistance, which is prevalent in most cancer cells.

Multifunctional particles for melanoma-targeted drug delivery.

New magnetic-based core-shell particles (MBCSPs) were developed to target skin cancer cells while delivering chemotherapeutic drugs in a controlled fashion. MBCSPs consist of a thermo-responsive shell of poly(N-isopropylacrylamide-acrylamide-allylamine) and a core of poly(lactic-co-glycolic acid) (PLGA) embedded with magnetite nanoparticles. To target melanoma cancer cells, MBCSPs were conjugated with Gly-Arg-Gly-Asp-Ser (GRGDS) peptides that specifically bind to the α(5)ß(3) receptors of melanoma cells. MBCSPs consist of unique multifunctional and controlled drug delivery characteristics. Specially, they can provide dual drug release mechanisms (a sustained release of drugs through degradation of PLGA core and a controlled release in response to changes in temperature via thermo-responsive polymer shell), and dual targeting mechanisms (magnetic localization and receptor-mediated targeting). Results from in vitro studies indicate that GRGDS-conjugated MBCSPs have an average diameter of 296 nm and exhibit no cytotoxicity towards human dermal fibroblasts up to 500 µg ml(-1). Further, a sustained release of curcumin from the core and a temperature-dependent release of doxorubicin from the shell of MBCSPs were observed. The particles also produced a dark contrast signal in magnetic resonance imaging. Finally, the particles were accumulated at the tumor site in a B16F10 melanoma orthotopic mouse model, especially in the presence of a magnet. Results indicate great potential of MBCSPs as a platform technology to target, treat and monitor melanoma for targeted drug delivery to reduce side effects of chemotherapeutic reagents.

Temperature-sensitive polymer-coated magnetic nanoparticles as a potential drug delivery system for targeted therapy of thyroid cancer.

The objective of this work was to develop and investigate temperature-sensitive poly(N-isopropylacrylamide-acrylamide-allylamine)-coated iron oxide magnetic nanoparticles (TPMNPs) as possible targeted drug carriers for treatments of advanced thyroid cancer (ATC). These nanoparticles were prepared by free radical polymerization of monomers on the surface of silane-coupled iron oxide nanoparticles. In vitro studies demonstrated that TPMNPs were cytocompatible and effectively taken up by cancer cells in a dose-dependent manner. An external magnetic field significantly increased nanoparticle uptake, especially when cells were exposed to physiological flow conditions. Drug loading and release studies using doxorubicin confirmed the temperature-responsive release of drugs from nanoparticles. In addition, doxorubicin-loaded nanoparticles significantly killed ATC cells when compared to free doxorubicin. The in vitro results indicate that TPMNPs have potential as targeted and controlled drug carriers for thyroid cancer treatment.

Multifunctionality of indocyanine green-loaded biodegradable nanoparticles for enhanced optical imaging and hyperthermia intervention of cancer.

The aim of this study was to develop and characterize multifunctional biodegradable and biocompatible poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with indocyanine green (ICG) as an optical-imaging contrast agent for cancer imaging and as a photothermal therapy agent for cancer treatment. PLGA-ICG nanoparticles (PIN) were synthesized with a particle diameter of 246±11 nm, a polydispersity index of 0.10±0.03, and ICG loading efficiency of 48.75±5.48%. PIN were optically characterized with peak excitation and emission at 765 and 810±5 nm, a fluorescence lifetime of 0.30±0.01 ns, and peak absorbance at 780 nm. The cytocompatibility study of PIN showed 85% cell viability till 1-mg/ml concentration of PIN. Successful cellular uptake of ligand conjugated PIN by prostate cancer cells (PC3) was also obtained. Both phantom-based and in vitro cell culture results demonstrated that PIN (1) have the great potential to induce local hyperthermia (i.e., temperature increase of 8 to 10°C) in tissue within 5 mm both in radius and in depth; (2) result in improved optical stability, excellent biocompatibility with healthy cells, and a great targeting capability; (3) have the ability to serve as an image contrast agent for deep-tissue imaging in diffuse optical tomography.