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The presence of immunosuppressive immune cells in cancer is a significant barrier to the generation of therapeutic immune responses. Similarly, in vivo triple-negative breast cancer (TNBC) models often contain prevalent tumor-associated macrophages in the tumor microenvironment (TME), resulting in breast cancer initiation, invasion, and metastasis by generating immunosuppressive environment. Here, we test systemic chemoimmunotherapy using small-molecule agents, paclitaxel (PTX), and colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397, to enhance the adaptive T cell immunity against TNBCs in immunocompetent mouse TNBC models. PTX and PLX3397 are very poorly soluble in water and shown poor therapeutic outcomes in TNBC animal models in conventional formulation. To address the challenge for the delivery of insoluble drugs to TNBC, we use high-capacity poly(2-oxazoline) (POx)-based polymeric micelles to greatly improve the solubility and widen the therapeutic index of such drugs. The results demonstrate that high-dose PTX in POx, even as a single agent, exerts strong effects on TME and induces the long-term immune memory. In addition, we demonstrate that the PTX and PLX3397 combination provides consistent therapeutic improvement across several TNBC models, resulting from the repolarization of the immunosuppressive TME and enhanced T cell immune response that suppress both the primary tumor growth and metastasis. Overall, the work emphasizes the benefit of drug reformulation and outlines potential translational path for both PTX and PTX with PLX3397 combination therapy using POx polymeric micelles for the treatment of TNBC.

The in vivo fate of nanoformulated drugs is governed by the physicochemical properties of the drug and the functionality of nanocarriers. Nanoformulations such as polymeric micelles, which physically encapsulate poorly soluble drugs, release their payload into the bloodstream during systemic circulation. This results in three distinct fractions of the drug-nanomedicine: encapsulated, protein-bound, and free drug. Having a thorough understanding of the pharmacokinetic (PK) profiles of each fraction is essential to elucidate mechanisms of nanomedicine-driven changes in drug exposure and PK/PD relationships pharmacodynamic activity. Here, we present a comprehensive preclinical assessment of the poly(2-oxazoline)-based polymeric micelle of paclitaxel (PTX) (POXOL hl-PM), including bioequivalence comparison to the clinically approved paclitaxel nanomedicine, Abraxane®. Physicochemical characterization and toxicity analysis of POXOL hl-PM was conducted using standardized protocols by the Nanotechnology Characterization Laboratory (NCL). The bioequivalence of POXOL hl-PM to Abraxane® was evaluated in rats and rhesus macaques using the NCL’s established stable isotope tracer ultrafiltration assay (SITUA) to delineate the plasma PK of each PTX fraction. The SITUA study revealed that POXOL hl-PM and Abraxane® had comparable PK profiles not only for total PTX but also for the distinct drug fractions, suggesting bioequivalence in given animal models. The comprehensive preclinical evaluation of POXOL hl-PM in this study showcases a series of widely-applicable standardized studies by NCL for assessing nanoformulations prior to clinical investigation.GRAPHICAL ABSTRACT

Significant advances in physicochemical properties of polymeric micelles enable optimization of therapeutic drug efficacy, supporting nanomedicine manufacturing and clinical translation. Yet, the effect of micelle morphology on pharmacological efficacy has not been adequately addressed. We addressed this gap by assessing pharmacological efficacy of polymeric micelles with spherical and worm-like morphologies. We observed that poly(2-oxazoline)-based polymeric micelles can be elongated over time from a spherical structure to worm-like structure, with elongation influenced by several conditions, including the amount and type of drug loaded into the micelles. We further evaluated the role of different morphologies of olaparib micelles on pharmacological performance against a triple-negative breast cancer tumor (TNBC) model. Spherical micelles accumulated rapidly in the tumor tissue while retaining large amounts of drug; worm-like micelles accumulated more slowly and only upon releasing significant amounts of drug. These findings suggest that the dynamic character of the drug–micelle structure and the micelle morphology play a critical role in pharmacological performance, and that spherical micelles are better suited for systemic delivery of anticancer drugs to tumors when drugs are loosely associated with the polymeric micelles.

CDK4/6 inhibitors hold promise for brain tumor treatment, but efficacy has been limited by recurrence in both preclinical models and clinical trials. To address recurrence, we tested a nanoparticle formulation of the CDK4/6 inhibitor palbociclib (POx-palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma. We then analyzed medulloblastomas in mice receiving palbociclib treatment, and compared the efficacy of combining palbociclib with specific inhibitors suggested by our analysis. POx-Palbo showed reduced toxicity compared to conventional palbociclib, was tolerable in parenteral administration, improved CNS pharmacokinetics, and extended survival of mice with medulloblastoma. Recurrence, however, remained problematic as fractions of tumor cells proliferated during therapy. ScRNA-seq identified a gene expression pattern unique to proliferating medulloblastoma cells in POx-Palbo-treated mice, marked by up-regulation of the glutamate transporterSlc1a2and down-regulation of diverse ribosomal genes. Reduced mTORC1 signaling, suggested by ribosomal suppression in POx-Palbo-treated tumors was confirmed by decreased 4EBP1 phosphorylation (p4EBP1). Further reducing mTORC1 activity by combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects, with increased suppression of both pRB and p4EBP1, and prolonged mouse survival compared to either agent alone. In contrast, targeting cell cycle progression by combining POx-Palbo with the SHH-pathway inhibitor vismodegib, or with the replication-targeting agents gemcitabine or etoposide, failed to enhance efficacy. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment, and identify POx-palbo plus sapanisertib as effective combinatorial therapy for SHH medulloblastoma. This combination may be appropriate for testing in patients with recurrence, who need new options.

Hypothesis Magnetic liposomes are shown to release the entrapped dye once modulated by low frequency AC MF. The mechanism and effectiveness of MF application should depend on lipid composition, magnetic nanoparticles (MNPs) properties, temperature and field parameters. Experiments The study was performed using liposomes of various lipid composition and embedded hydrophobic MNPs. The liposomes structural changes were studied by the transmission electron microscopy (TEM) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and the leakage was monitored by the fluorescent dye release. Findings Magnetic liposomes exposure to the AC MF resulted in the clustering of the MNPs in the membranes and disruption of the lipid packaging. Addition of cholesterol diminished the dye release from the saturated lipid-based liposomes. Replacement of the saturated lipid for unsaturated one also decreased the dye release. The dye release depended on the strength, but not the frequency of the field. Thus, the oscillating motion of MNPs in AC MF ruptures the gel phase membranes of saturated lipids. As the temperature increases the disruption also increases. In the liquid crystalline membranes formed by unsaturated lipids the deformations and defects created by mechanical motion of the MNPs are more likely to heal and results in decreased release.

Cancer immunotherapy, the utilization of the patients’ own immune system to treat cancer, has emerged as a powerful new strategy in cancer treatment. Recent clinical data has demonstrated immunotherapy to be effective in a wide range of cancers, including lung, bladder, renal cell, colorectal, gastro-esophageal, and head and neck cancers5-11. However, clinical reports of immune-based treatments for medulloblastomas are scarce and preliminary. Therefore, there is a need in developing strategies to improve medulloblastoma immunotherapy. Our recent studies have confirmed that SHH medulloblastomas are enriched in Tumor Associated Macrophages (TAMs) and unlike other tumors, TAMs are associated with positive outcomes and play a positive role by impairing tumor growth. Overall, analysis of TME in medulloblastomas reveals TAMs as a potential therapeutic target. Resiquimod is a synthetic small molecule agonist of Toll-like receptors 7 and 8 (TLR7/8) that modulates innate immune cells. We have loaded resiquimod into ultra-high-capacity polyoxazoline (POx) block copolymers forming small, homogeneous nanoparticles (POx-resiquimod). Our recent study shows that loading into POx nanoparticles improves drug delivery to tumors and treatment with 3 injections of POx-res as the single-agent treatment results in a profound anti-tumor effect G-Smo mice while treatment with free drug shows no therapeutic benefit. Our data also shows that that the tumors of G-Smo mice are enriched with the mixed populations of anti-inflammatory and pro-inflammatory macrophages and the treatment with POx-resiquimod have enhanced infiltration of macrophages into the tumors, enhanced the repolarization of macrophages to M1 subtype and decreased tumor cells viability. These studies show for the first time that targeting the medulloblastoma TME with POx-resiquimod can produce a significant anti-tumor effect. Furthermore, combination of POx-resiquimod with radiation resulted in long term survivals, showing potential therapeutic combination. The expression of TLR7/8 on TAMs in patient-derived medulloblastoma samples suggests that resiquimod may produce similar anti-medulloblastoma effects in humans.

Many new drug development candidates are highly lipophilic compounds with low water solubility. This constitutes a formidable challenge for the use of such compounds for cancer therapy, where high doses and intravenous injections are needed ( Di et al., 2012 ). Here, we present a poly(2-oxazoline) polymer (POx)-based nanoformulation strategy to solubilize and deliver hydrophobic drugs. POx micelles are prepared by a simple thin-film hydration method. In this method, the drug and polymer are dissolved in a common solvent and allowed to mix, following which the solvent is evaporated using mild heating conditions to form a thin film. The micelles form spontaneously upon hydration with saline. POx nanoformulation of hydrophobic drugs is unique in that it has a high drug loading capacity, which is superior to micelles of conventional surfactants. Moreover, multiple active pharmaceutical ingredients (APIs) can be included within the same POx micelle, thereby enabling the codelivery of binary as well as ternary drug combinations ( Han et al., 2012 ; He et al., 2016 ).

Macrophages are desirable targets for gene therapy of cancer and other diseases. Cationic diblock copolymers of polyethylene glycol (PEG) and poly-L-lysine (PLL) or poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (pAsp(DET)) are synthesized and used to form polyplexes with a plasmid DNA (pDNA) that are decorated with mannose moieties, serving as the targeting ligands for the C type lectin receptors displayed at the surface of macrophages. The PEG-b-PLL copolymers are known for its cytotoxicity, so PEG-b-PLL-based polyplexes are cross-linked using reducible reagent dithiobis(succinimidyl propionate) (DSP). The cross-linked polyplexes display low toxicity to both mouse embryonic fibroblasts NIH/3T3 cell line and mouse bone marrow-derived macrophages (BMMΦ). In macrophages mannose-decorated polyplexes demonstrate an ≈8 times higher transfection efficiency. The cross-linking of the polyplexes decrease the toxicity, but the transfection enhancement is moderate. The PEG-b-pAsp(DET) copolymers display low toxicity with respect to the IC-21 murine macrophage cell line and are used for the production of non-cross-linked pDNA-contained polyplexes. The obtained mannose modified polyplexes exhibit ca. 500-times greater transfection activity in IC-21 macrophages compared to the mannose-free polyplexes. This result greatly exceeds the targeting gene transfer effects previously described using mannose receptor targeted non-viral gene delivery systems. These results suggest that Man-PEG-b-pAsp(DET)/pDNA polyplex is a potential vector for immune cells-based gene therapy.

Tumor xenograft models developed by transplanting human tissues or cells into immune-deficient mice are widely used to study human cancer response to drug candidates. However, immune-deficient mice are unfit for investigating the effect of immunotherapeutic agents on the host immune response to cancer (Morgan, 2012). Here, we describe the preparation of an orthotopic, syngeneic model of lung adenocarcinoma (LUAD), a subtype of non-small cell lung cancer (NSCLC), to study the antitumor effect of chemo and immunotherapeutic agents in an immune-competent animal. The tumor model is developed by implanting 344SQ LUAD cells derived from the metastases of Kras(G12D); p53(R172HΔG )genetically engineered mouse model into the left lung of a syngeneic host (Sv/129). The 344SQ LUAD model offers several advantages over other models: 1) The immune-competent host allows for the assessment of the biologic effects of immune-modulating agents; 2) The pathophysiological features of the human disease are preserved due to the orthotopic approach; 3) Predisposition of the tumor to metastasize facilitates the study of therapeutic effects on primary tumor as well as the metastases ( Chen et al., 2014 ). Furthermore, we also describe a treatment strategy based on Poly(2-oxazoline) micelles that has been shown to be effective in this difficult-to-treat tumor model ( Vinod et al., 2020b ).

Polymeric micelle formulation of an immune modulator improves survival in a highly aggressive model of non–small cell lung cancer., About 40% of patients with non–small cell lung cancer (NSCLC) have stage IV cancer at the time of diagnosis. The only viable treatment options for metastatic disease are systemic chemotherapy and immunotherapy. Nonetheless, chemoresistance remains a major cause of chemotherapy failure. New immunotherapeutic modalities such as anti–PD-1 immune checkpoint blockade have shown promise; however, response to such strategies is highly variable across patients. Here, we show that our unique poly(2-oxazoline)–based nanomicellar formulation (PM) of Resiquimod, an imidazoquinoline Toll-like receptor (TLR) 7/8 agonist, had a superior tumor inhibitory effect in a metastatic model of lung adenocarcinoma, relative to anti–PD-1 therapy or platinum-based chemotherapy. Investigation of the in vivo immune status following Resiquimod PM treatment showed that Resiquimod-based stimulation of antigen-presenting cells in the tumor microenvironment resulted in the mobilization of an antitumor CD8+ immune response. Our study demonstrates the promise of poly(2-oxazoline)-formulated Resiquimod for treating metastatic NSCLC.

We report a novel, nanoparticle formulation of the SHH pathway inhibitor vismodegib that improves efficacy for medulloblastoma treatment while reducing toxicity. Systemic therapies for brain tumors are complicated by restricted blood-brain barrier (BBB) permeability and dose-limiting extraneural toxicity, therefore improved delivery approached are needed. Here we show how a nanoparticle delivery system addresses these obstacles, bringing new efficacy to previously ineffective therapy. Vismodegib has been a promising agent for patients with SHH- subgroup medulloblastoma and is FDA-approved for basal cell carcinoma. However, vismodegib has limited benefit for patients with SHH-driven medulloblastoma, due to off-target toxicities and the development of resistance during therapy. We encapsulated vismodegib in polyoxazoline block copolymer micelles (POx-vismo). We then evaluated POx-vismo using transgenic mice engineered to develop endogenous medulloblastomas, testing the novel agent in a preclinical model with native vasculature and tumor microenvironment. POx-vismo showed improved CNS pharmacokinetics and reduced systemic and bone toxicity. Mechanistic studies show that POx nanoparticles did not enter the CNS, but rather acted within the vascular compartment to improve drug delivery by decreasing drug binding to serum proteins and reducing the volume of distribution. POx-vismo demonstrated improved efficacy, extending the survival of medulloblastoma-bearing mice. Our results show the potential for a simple, non-targeted nanoparticle formulation to improve systemic brain tumor therapy, and specifically to enhance vismodegib therapy for SHH-driven cancers.

Remote nano-magneto-mechanical actuation of magnetic nanoparticles (MNPs) by non-heating extremely low frequency magnetic field (ELF MF) is explored as a tool for non-invasive modification of bionanomaterials in pharmaceutical and medical applications. Here we study the effects of ELF MF (30–160 Hz, 8–120 kA/m) on the activity and release of a model enzyme, superoxide dismutase 1 (SOD1) immobilized by polyion coupling on dispersed MNPs aggregates coated with poly(L-lysine)-block-poly(ethylene glycol) block copolymer (s-MNPs). Such fields do not cause any considerable heating of MNPs but promote their rotating-oscillating mechanical motion that produces mechanical forces and deformations in adjacent materials. We observed the changes in the catalytic activity of immobilized SOD1 as well as its release from the s-MNPs/SOD1 polyion complex upon application of the ELF MF for 5 to 15 min. At longer exposures (25 min) the s-MNPs/SOD1 dispersion destabilizes. The bell-shaped effect of the field frequency with maximum at f = 50 Hz and saturation effect of field strength (between 30 kA/m and 120 kA/m at f = 50 Hz) are reported and explained. The findings are significant as one early indication of the nano-magneto-mechanical disruption by ELF MF of cooperative polyion complexes that are widely used for design of current functional healthcare bionanomaterials.

Combining diagnosis and treatment approaches in one entity is the goal of theranostics for cancer therapy. Magnetic nanoparticles have been extensively used as contrast agents for nuclear magnetic resonance imaging as well as drug carriers and remote actuation agents. Poly(2-oxazoline)-based polymeric micelles, which have been shown to efficiently solubilize hydrophobic drugs and drug combinations, have high loading capacity (above 40% w/w) for paclitaxel. In this study, we report the development of novel theranostic system, NanoFerrogels, which is designed to capitalize on the magnetic nanoparticle properties as imaging agents and the poly(2-oxazoline)-based micelles as drug loading compartment. We developed six formulations with magnetic nanoparticle content of 0.3%-12% (w/w), with the z-average sizes of 85-130 nm and ξ-potential of 2.7-28.3 mV. The release profiles of paclitaxel from NanoFerrogels were notably dependent on the degree of dopamine grafting on poly(2-oxazoline)-based micelles. Paclitaxel loaded NanoFerrogels showed efficacy against three breast cancer lines which was comparable to free paclitaxel. They also showed improved tumor and lymph node accumulation and signal reduction in vivo (2.7% in tumor; 8.5% in lymph node) compared to clinically approved imaging agent ferumoxytol (FERAHEME®) 24 h after administration. NanoFerrogels responded to super-low frequency alternating current magnetic field (50 kA m−1, 50 Hz) which accelerated drug release from paclitaxel-loaded NanoFerrogels or caused death of cells loaded with NanoFerrogels. These proof-of-concept experiments demonstrate that NanoFerrogels have potential as remotely actuated theranostic platform for cancer diagnosis and treatment.

About 40% of the NSCLC patients have Stage IV cancer at the time of diagnosis. The only viable treatment options for metastatic disease are systemic chemotherapy and immunotherapy. Nonetheless, chemoresistance remains a major cause of chemotherapy failure. New immunotherapeutic modalities such as anti-PD1 checkpoint blockade have shown promise; however, response to such strategies is highly variable across patients. Here, we show that our novel poly(2-oxazoline) (POx) based nanomicellar formulation of Resiquimod, an imidazoquinoline TLR 7/8 agonist, had a superior tumor inhibitory effect in a metastatic model of lung adenocarcinoma, relative to anti-PD1 immune checkpoint blockade therapy as well as platinum-based chemotherapy, which is the mainstay of treatment for NSCLC. Investigation of the in vivo immune status following Resiquimod PM (POx micellar formulation of Resiquimod) treatment showed that Resiquimod-based stimulation of antigen-presenting cells in the tumor microenvironment resulted in the mobilization of anti-tumor CD8+immune response. Our study demonstrates the promise of optimally delivered and nano-formulated Resiquimod as a new immunomodulating therapeutic strategy for the treatment of metastatic NSCLC.

Magnetomechanical modulation of biochemical processes is a promising instrument for bioengineering and nanomedicine. This work demonstrates two approaches to control activity of an enzyme, α-chymotrypsin immobilized on the surface of gold-coated magnetite magnetic nanoparticles (GM-MNPs) using a nonheating low-frequency magnetic field (LF MF). The measurement of the enzyme reaction rate was carried out in situ during exposure to the magnetic field. The first approach involves α-chymotrypsin-GM-MNPs conjugates, in which the enzyme undergoes mechanical deformations with the reorientation of the MNPs under LF MF (16-410 Hz frequency, 88 mT flux density). Such mechanical deformations result in conformational changes in α-chymotrypsin structure, as confirmed by infrared spectroscopy and molecular modeling, and lead to a 63% decrease of enzyme initial activity. The second approach involves an α-chymotrypsin-GM-MNPs/trypsin inhibitor-GM-MNPs complex, in which the activity of the enzyme is partially inhibited. In this case the reorientation of MNPs in the field leads to disruption of the enzyme-inhibitor complex and an almost 2-fold increase of enzyme activity. The results further demonstrate the utility of magnetomechanical actuation at the nanoscale for the remote modulation of biochemical reactions.

Significant advances in physicochemical properties of polymeric micelles enable optimization of therapeutic drug efficacy, supporting nanomedicine manufacturing and clinical translation. Yet, the effect of micelle morphology on pharmacological efficacy is not adequately addressed. This work addresses this gap by assessing pharmacological efficacy of polymeric micelles with spherical and worm-like morphologies. It is observed that poly(2-oxazoline)-based polymeric micelles can be elongated over time from a spherical structure to worm-like structure, with elongation influenced by several conditions, including the amount and type of drug loaded into the micelles. The role of different morphologies on pharmacological performance of drug loaded micelles against triple-negative breast cancer and pancreatic cancer tumor models is further evaluated. Spherical micelles accumulate rapidly in the tumor tissue while retaining large amounts of drug; worm-like micelles accumulate more slowly and only upon releasing significant amounts of drug. These findings suggest that the dynamic character of the drug-micelle structure and the micelle morphology play a critical role in pharmacological performance, and that spherical micelles are better suited for systemic delivery of anticancer drugs to tumors when drugs are loosely associated with the polymeric micelles.

The in vivo fate of nanoformulated drugs is governed by the physicochemical properties of the drug and the functionality of nanocarriers. Nanoformulations such as polymeric micelles, which physically encapsulate poorly soluble drugs, release their payload into the bloodstream during systemic circulation. This results in three distinct fractions of the drug-nanomedicine: encapsulated, protein-bound, and free drug. Having a thorough understanding of the pharmacokinetic (PK) profiles of each fraction is essential to elucidate mechanisms of nanomedicine-driven changes in drug exposure and PK/PD relationships pharmacodynamic activity. Here, we present a comprehensive preclinical assessment of the poly (2-oxazoline)-based polymeric micelle of paclitaxel (PTX) (POXOL hl-PM), including bioequivalence comparison to the clinically approved paclitaxel nanomedicine, Abraxane®. Physicochemical characterization and toxicity analysis of POXOL hl-PM was conducted using standardized protocols by the Nanotechnology Characterization Laboratory (NCL). The bioequivalence of POXOL hl-PM to Abraxane® was evaluated in rats and rhesus macaques using the NCL's established stable isotope tracer ultrafiltration assay (SITUA) to delineate the plasma PK of each PTX fraction. The SITUA study revealed that POXOL hl-PM and Abraxane® had comparable PK profiles not only for total PTX but also for the distinct drug fractions, suggesting bioequivalence in given animal models. The comprehensive preclinical evaluation of POXOL hl-PM in this study showcases a series of widely applicable standardized studies by NCL for assessing nanoformulations prior to clinical investigation.

Systemic therapies for brain tumors are complicated by dose-limiting extraneural toxicity, serum protein binding and restricted blood-brain barrier (BBB) permeability. For patients with SHH-subgroup medulloblastoma, SHH-pathway inhibition may be more effective and less toxic than current non-targeted therapy. However, vismodegib, which effectively targets SHH-driven basal cell carcinoma, has limited benefit for patients with SHH-driven medulloblastoma. Formulating vismodegib in poly(2-oxazoline) micelles (POx-vismo) improved delivery and efficacy, using medulloblastoma-prone hGFAP-Cre/SmoM2 (G-Smo) mice for a preclinical model. Compared to conventional vismodegib (c-vismo), POx-vismo increased maximum tolerated dose (MTD), tumor:serum ratio and penetration to brain and tumor. Further studies have shown the ability of POx micelles to load CDK4/6 inhibitor. Loading into POx micelles improved MTD and efficacy of palbociclib. Taken together, our results show the potential for poly(2-oxazoline) micelles delivery to make failed brain tumor treatments newly effective. This work was supported by the NCI Alliance for Nanotechnology in Cancer (U54CA198999, Carolina Center of Cancer Nanotechnology Excellence), by NINDS (R01NS088219, R01NS102627) and by the St. Baldrick’s Foundation. References: 1 H. Bader et al. Angew. Macromol. Chem. 1984, 123/124:457; A. Kabanov et al. FEBS Lett. 1989, 258:343; M. Yokoyama et al. Cancer Res. 1990, 50:1693. 2 M. Yokoyama et al. J. Exp. Clin. Med. 2011, 3:8. 3 T. Lorson et al. Biomaterials. 2018, 178:204. 4 R. Luxenhofer et al. Biomaterials 2010, 31:4972; A. Schulz et al. ACS Nano 2014, 8 (3):2686; Z. He et al. Biomaterials 2016, 101:296

The tendency of brain cells to undergo apoptosis in response to exogenous events varies across neural development, with apoptotic threshold dependent on proliferation state. Proliferative neural progenitors show a low threshold for apoptosis, while terminally differentiated neurons are relatively refractory. To define the mechanisms linking proliferation and apoptotic threshold, we examined the effect of conditionally deleting Bcl2l1, the gene that codes the antiapoptotic protein BCL-xL, in cerebellar granule neuron progenitors (CGNPs), and of co-deleting Bcl2l1 homologs, antiapoptotic Mcl-1, or pro-apoptotic Bax. We found that cerebella in conditional Bcl2l1-deleted (Bcl-xL(cKO)) mice were severely hypoplastic due to the increased apoptosis of CGNPs and their differentiated progeny, the cerebellar granule neurons (CGNs). Apoptosis was highest as Bcl-xL(cKO) CGNPs exited the cell cycle to initiate differentiation, with proliferating Bcl-xL(cKO) CGNPs relatively less affected. Despite the overall reduction in cerebellar growth, SHH-dependent proliferation was prolonged in Bcl-xL(cKO) mice, as more CGNPs remained proliferative in the second postnatal week. Co-deletion of Bax rescued the Bcl-xL(cKO) phenotype, while co-deletion of Mcl-1 enhanced the phenotype. These findings show that CGNPs require BCL-xL to regulate BAX-dependent apoptosis, and that this role can be partially compensated by MCL-1. Our data further show that BCL-xL expression regulates MCL-1 abundance in CGNPs, and suggest that excessive MCL-1 in Bcl-xL(cKO) mice prolongs CGNP proliferation by binding SUFU, resulting in increased SHH pathway activation. Accordingly, we propose that BCL-xL and MCL-1 interact with each other and with developmental mechanisms that regulate proliferation, to adjust the apoptotic threshold as CGNPs progress through postnatal neurogenesis to CGNs.

Computer-assisted strategy for drug delivery leads to discovery of polymeric micelle formulations for poorly soluble drugs., Many drug candidates fail therapeutic development because of poor aqueous solubility. We have conceived a computer-aided strategy to enable polymeric micelle-based delivery of poorly soluble drugs. We built models predicting both drug loading efficiency (LE) and loading capacity (LC) using novel descriptors of drug-polymer complexes. These models were employed for virtual screening of drug libraries, and eight drugs predicted to have either high LE and high LC or low LE and low LC were selected. Three putative positives, as well as three putative negative hits, were confirmed experimentally (implying 75% prediction accuracy). Fortuitously, simvastatin, a putative negative hit, was found to have the desired micelle solubility. Podophyllotoxin and simvastatin (LE of 95% and 87% and LC of 43% and 41%, respectively) were among the top five polymeric micelle-soluble compounds ever studied experimentally. The success of the strategy described herein suggests its broad utility for designing drug delivery systems.

Here we report a novel poly(2-oxazoline)-based block copolymer with the aromatic heterocyclic side chains in one block, poly(2-methyl-2-oxazoline)-b-poly(2-N,N-dimethyl-1,3,5-triazine-2,4-diamine-6-ethyl-2-oxazoline) (PMeOx-PcBOx), and demonstrate its potential application as a drug delivery platform. The copolymer was synthesized via the condensation of N,N-dimethylbiguanide with the methyl ester side chain in poly(2-methoxycarboxyethyl-2-oxazoline) block (PMestOx) of the PMeOx-PMestOx diblock copolymer. We confirmed the N,N-dimethylbiguanide condensation with PMestOx and the complete conversion of the side chain to the N,N-dimethyl-1,3,5-triazine-2,4-diamine-6-ethyl moiety by NMR spectroscopy, MALDI-TOF mass spectroscopy, UV-Vis spectroscopy, and titration analysis. The PMeOx-PcBOx copolymer self-assemble into polymeric micelles in aqueous solution. Successful encapsulation into these micelles has been demonstrated for 1) several poorly soluble drugs, such as bruceantin and LY2109761, and 2) dichloro(1,2-diaminocyclohexane)platinum(II) (DachPt). The first class of drugs is incorporated possibly via hydrogen bonding and pi-pi interactions with the PcBOx side groups, while the second one is likely forms coordination bonds with the same side groups. The capability of this new copolymer to solubilize a uniquely diverse set of active pharmaceutical ingredients suggests potential applications in drug delivery.

Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. We report that ATR, which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. Our data show that p53-driven apoptosis and cell cycle arrest – and, in the absence of p53, non-apoptotic cell death – redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition.

Given the superior soft tissue contrasts obtained by MRI and the long residence times of magnetic nanoparticles (MNPs) in soft tissues, MNP-based theranostic systems are being developed for simultaneous imaging and treatment. However, development of such theranostic nanoformulations presents significant challenges of balancing the therapeutic and diagnostic functionalities in order to achieve optimum effect from both. Here we developed a simple theranostic nanoformulation based on magnetic nanoclusters (MNCs) stabilized by a bisphosphonate-modified poly(glutamic acid)-b-(ethylene glycol) block copolymer and complexed with cisplatin. The MNCs were decorated with luteinizing hormone releasing hormone (LHRH) to target LHRH receptors (LHRHr) overexpressed in ovarian cancer cells. The targeted MNCs significantly improved the uptake of the drug in cancer cells and decreased its IC(50) compared to the nontargeted formulations. Also, the enhanced LHRHr-mediated uptake of the targeted MNCs resulted in enhancement in the T(2)-weighted negative contrast in cellular phantom gels. Taken together, the LHRH-conjugated MNCs show good potential as ovarian cancer theranostics.

For the roughly 30% of medulloblastoma patients with SHH-subgroup tumors, drugs that disrupt SHH signaling offer the potential of improved efficacy with reduced toxicity compared to current, non-targeted treatment. The FDA-approved SMO inhibitor vismodegib, has been shown to be safe and effective for the treatment of basal cell carcinoma. In SHH-subgroup medulloblastomas, however, initial responses to vismodegib, have been followed by resistance that develops during treatment. We hypothesized that improving the delivery of the drug across the blood-brain barrier would forestall the development of resistance. We developed a novel, nanoparticle formulation of vismodegib, encapsulated in polyoxazoline micelles (POx-vismo). These nanoparticles showed high drug loading, and consistent, nanometer-scale particle size. We treated medulloblastoma bearing GFAP-cre/SmoM2 (G-Smo) mice with either POx-vismo or conventional vismodegib, administered by IP injection at postnatal days 12 and 13 (P12, P13) and then every other day. Both formulations induced an initial response. However, G-Smo mice treated with conventional vismodegib quickly progressed despite therapy. In contrast, POx-vismo-treated mice survived significantly longer than untreated mice or mice treated with conventional vismodegib. Our results show the superiority of POx-vismo and highlight the potential for POx micelles drug delivery to increase the efficacy of approved and experimental agents for brain tumor therapy. The anti-tumor effect of vismodegib, optimized in POx-vismo is significant and may, in combination with additional treatments such as radiation, lead to new treatment approaches.

Superabsorbent sodium polyacrylate polymeric hydrogels that retain large amounts of liquids are used in disposable diapers, sanitary napkins, and other applications. These polymers are generally considered “nontoxic” with acute oral median lethal doses (LD50) >5 g/kg. Despite this favorable toxicity profile, we identified a novel toxic syndrome in dogs and rats following the ingestion of a commercial dog pad composed primarily of a polyacrylic acid hydrogel. Inappropriate mentation, cerebellar ataxia, vomiting, and intention tremors were observed within 24 h after the ingestion of up to 15.7 g/kg of the hydrogel by an adult, castrated male Australian Shepherd mix. These observations prompted an experimental study in rats to further characterize the toxicity of the hydrogel. Adult, female Sprague Dawley rats ( n = 9) were assessed before and after hydrogel ingestion (2.6–19.2 g/kg over 4 h) using a functional observation battery and spontaneous motor activity. Clinical signs consistent with neurotoxicity emerged in rats as early as 2 h after the end of hydrogel exposure, including decreased activity in an open field, hunched posture, gait changes, reduced reaction to handling, decreased muscle tone, and abnormal surface righting. Hydrogel-exposed rats also had reduced motor activity when compared with pre-exposure baseline data. Rats that ingested the hydrogel did not develop nervous system lesions. These findings support the conclusion that some pet pad hydrogel products can induce acute neurotoxicity in animals under high-dose exposure conditions.

Nanoparticle-based systems for concurrent delivery of multiple drugs can improve outcomes of cancer treatments, but face challenges because of differential solubility and fairly low threshold for incorporation of many drugs. Here we demonstrate that this approach can be used to greatly improve the treatment outcomes of etoposide (ETO) and platinum drug combination ("EP/PE") therapy that is the backbone for treatment of prevalent and deadly small cell lung cancer (SCLC). A polymeric micelle system based on amphiphilic block copolymer poly(2-oxazoline)s (POx) poly(2-methyl-2-oxazoline- block-2-butyl-2-oxazoline- block-2-methyl-2-oxazoline) (P(MeOx- b-BuOx- b-MeOx) is used along with an alkylated cisplatin prodrug to enable co-formulation of EP/PE in a single high-capacity vehicle. A broad range of drug mixing ratios and exceptionally high two-drug loading of over 50% wt. drug in dispersed phase is demonstrated. The highly loaded POx micelles have worm-like morphology, unprecedented for drug loaded polymeric micelles reported so far, which usually form spheres upon drug loading. The drugs co-loading in the micelles result in a slowed-down release, improved pharmacokinetics, and increased tumor distribution of both drugs. A superior antitumor activity of co-loaded EP/PE drug micelles compared to single drug micelles or their combination as well as free drug combination was demonstrated using several animal models of SCLC and non-small cell lung cancer.

This work is focused on synthesis and characterization of targeted magnetic nanoparticles as magnetic resonance imaging (МRI) agents for in vivo visualization of gliomas. Ferric oxide (Fe 3 O 4 ) cores were synthesized by thermal decomposition and coated with bovine serum albumin (BSA) to form nanoparticles with D eff of 53±9nm. The BSA was further cross-linked to improve colloidal stability. Monoclonal antibodies against vascular endothelial growth factor (mAbVEGF) were covalently conjugated to BSA through a polyethyleneglycol linker. Here we demonstrate that 1) BSA coated nanoparticles are stable and non-toxic to different cells at concentration up to 2.5mg/mL; 2) conjugation of monoclonal antibodies to nanoparticles promotes their binding to VEGF-positive glioma С6 cells in vitro ; 3) targeted nanoparticles are effective in MRI visualization of the intracranial glioma. Thus, mAbVEGF-targeted BSA-coated magnetic nanoparticles are promising MRI contrast agents for glioma visualization. From the Clinical Editor This work focuses on synthesis and characterization of targeted magnetic nanoparticles as magnetic resonance imaging (МRI) agents for in vivo visualization of gliomas. The authors utilize the fact that high-grade gliomas have extensive areas of necrosis and hypoxia, which results in increased secretion of angiogenesis vascular endothelial growth factor (VEGF). Monoclonal antibodies against vascular endothelial growth factor (mAbVEGF) were covalently conjugated to crosslinked BSA coated ferric oxide (Fe3O4) nanoparticles. The results show that these targeted nanoparticles are effective in MRI visualization of the intracranial glioma and may provide a new and promising contrast agent.

The possibility of remotely inducing a defined effect on NPs by means of electromagnetic radiation appears attractive. From a practical point of view, this effect opens horizons for remote control of drug release systems, as well as modulation of biochemical functions in cells. Gold-coated magnetite nanoparticles are perfect candidates for such application. Herein, we have successfully synthesized core–shell NPs having magnetite cores and gold shells modified with various sulphur containing ligands and developed a new, simple and robust procedure for the purification of the resulting nanoparticles. The carboxylic groups displayed at the surface of the NPs were utilized for NP conjugation with a model enzyme (ChT). In the present study, we report the effect of the low-frequency AC magnetic field on the catalytic activity of the immobilized ChT. We show that the enzyme activity decreases upon exposure of the NPs to the field.

Site-directed administration of local anesthetic agents incorporated into a slow controlled-release injectable implant prolongs the analgesic effect. However, there are potential neuro- and myotoxic consequences. We evaluated a local anesthetic agent (bupivacaine) loaded into a slow-release biodegradable polymer based on castor oil and poly(lactic acid). The formulation was applied directly to the sciatic nerve area in female imprinting control region mice along with appropriate controls. Local nerve and muscle and systemic toxicity were evaluated over a 3-month period following injection of 0.05, 0.1, and 0.125 mL of the 15% bupivacaine–polymer formulation. Histological samples were prepared and examined; no signs of severe inflammation were observed. Histological inflammation signs were more prominent in both nerves and muscles following application of the largest volumes of the polymer formulation (0.1 and 0.125 mL). Following application of 0.1 mL, 15% bupivacaine–polymer formulation, maximal changes were seen in nerve samples two days and two weeks after injection, with complete resolution one month following injection. Neither blank polymer nor plain bupivacaine 0.5% caused any histological changes. Local nerve and muscle toxicity were affected by duration the of exposure and dose of the local anesthetic agent. However, there were clear indications of time-related healing process 3 months after injection.

Synthesis of gold nanoparticles (GNP) by reacting chloroauric acid (HAuCl4) and Pluronic F127 was thoroughly investigated. The rate of reduction of HAuCl4 and the yield and morphology of GNP strongly depended on the concentration of the reactants and sodium chloride, as well as pH and temperature. Upon completion of the reaction heterogeneous mixtures of small GNP of defined shape and Pluronic aggregates were formed. GNP were separated from the excess of Pluronic by centrifugal filtration. Under optimized conditions the GNP were small (ca. 80 nm), uniform (PDI ~0.09), strongly negatively charged (&zeta, potential &minus, 30 mV) and nearly spherical. They were stable in distilled water and phosphate-buffered saline. Purified GNP contained ~13% by weight of an organic component, yet presence of polypropylene oxide was not detected suggesting that Pluronic was not adsorbed on their surface. Analysis of the soluble products suggested that the copolymer undergoes partial degradation accompanied by cleavage of the C&ndash, O bonds and appearance of new primary hydroxyl groups. The reaction involves formation of free radicals and hydroperoxides depends on the oxygen concentration. GNP did not form at 4 &deg, C when the micellization of Pluronic was abolished reinforcing the role of the copolymer self-assembly. In conclusion, this work provides insight into the mechanism of HAuCl4 reduction and GNP formation in the presence of Pluronic block copolymers. It is useful for improving the methods of manufacturing uniform and pure GNP that are needed as nanoscale building blocks in nanomedicine applications.

It is proposed to use single-domain rodlike magnetic nanoparticles (MNPs) as mediators for nanomechanical control of properties of biological membranes and cells on the molecular or cellular level by exposing them to a homogeneous nonheating low-frequency magnetic field (AC MF). The trigger effect is achieved due to rotatory-oscillatory motion of MNPs in the AC MF, which causes the needed deformations in macromolecules of the membrane interacting with these MNPs.

The dynamics of single-domain magnetic nanoparticles cross-linked into multiparticle aggregates by organic ligands is considered. Mechanical factors of the effect of low frequency magnetic field on macromolecules attached to magnetic nanoparticles/aggregates within a suspension or gel are analyzed. The optimum conditions ensuring the best control over biochemical reactions in suspension by an external magnetic field (i.e., the ranges of frequency and magnetic field intensities, and the size of magnetic nanoparticles and shells covering them) are determined.

Weak magnetic fields (< 1 T) are known to change the paths and kinetics of some radical chemical reactions at room temperature through spin conversion of short-lived radical pairs. [1] Magnetic fields were also used to tune enzyme activity (oxidation of glucose). Specifically, exposure of magnetic nanoparticles functionalized with enzymes to magnetic field allowed the movement of particles in proximity to electrode, thus activating the enzyme. [2] Moreover, radio-frequency (RF) alternating current (AC) magnetic fields can induce the heating of single-domain magnetic nanoparticles (MNPs), and this can be used in magnetic hyperthermia to kill tumors. [3] Herein we describe a distinct effect of non-heating superlow-frequency magnetic fields as well as more conventional RF magnetic fields on the kinetics of chemical reactions catalyzed by the enzymes a-chymotrypsin (ChT) or bgalactosidase (b-GaL) immobilized on nanoscale MNP aggregates. Magnetite MNPs (7 to 12 nm diameter) were synthesized by reducing tris(acetylacetonato)iron(III), then they were coated with anionic poly(ethylene glycol)-bpolyacrylate (PEG-PAA), or poly(ethylene glycol)-b-polymethacrylate (PEG-PMA) copolymers. The polymers were bound to the magnetite surfaces by the carboxylate groups. [4] According to thermogravimetric analysis, the content of Fe3O4 in both coated MNP materials was about 35 wt %, and this was in excellent agreement with that measured by inductively coupled plasma-mass spectrometry. Upon dispersion in water (pH 6.5), they formed nanoscale, negatively charged aggregates (hydrodynamic intensity average diameters and zeta potentials of 194 nm, 70 mV and 39 nm, 49 mV for PEG-PAA/MNP and PEG-PMA/MNP, respectively, as determined by dynamic light scattering). Based on TEM, they contained clusters of 7 to 20 MNP grains. Enzymes were conjugated to these aggregates using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide and N-hydroxysulfosuccinimide to couple amines on the protein to carboxylic acids on the copolymer (Scheme 1).

Prolonged analgesia may be achieved using a single injection of slow-release local anesthetic formulation. The study objective was to improve the efficacy of a previously reported formulation comprising 10% bupivacaine in poly(DL:lactic acid co castor oil) 3:7. The polymer was loaded with 15% bupivacaine and injected through a 22G needle close to the sciatic nerve of ICR mice. Sensory and motor nerve blockade were measured. The efficacy and toxicity of the polymer-drug combination were determined. Sixty percent of the incorporated bupivacaine was released during 1 week in vitro. During in vitro release no burst effect was seen, suggesting low toxicity of the formulation. Single injection of 0.1 mL of 15% polymer-bupivacaine formulation caused motor block that lasted 64 h and sensory block that lasted 96 h. The MTD of the polymer-drug formulation was established as 0.175 mL. Microscopic examination of the injection sites revealed reversible nerve inflammation and normal internal organs. The polymer poly(DL:lactic acid co castor oil) 3:7 is a safe carrier for prolonged activity of bupivacaine up to 96 h. The increase of drug load in the formulation reduces the drug release rates due to stronger polymer-drug interactions and higher overall hydrophobicity of the formulation.

A systematic study on the influence of lactic acid (LA) conformation on polyester properties, degradation, and drug release profiles is reported. Copolyesters based on L- or DL-LA and castor oil (CO) (triglyceride of ricinoleic acid (RA)) were synthesized by melt condensation. Two feed ratios of LA to CO were used. Copolymers with feed ratio of 60:40 and 50:50 LA to CO were synthesized. P(LA/CO)s 60:40 and 50:50 had weight average molecular weights (Mw) of 4700 and 3700, respectively. All polymers are viscous liquids at room temperature. Copolymer composition and LA conformation had direct effect on polymer properties, degradation rate, and drug release profile. Increase in CO content (hydrophobisity) decreased the degradation and release rates of the polymers. Effect of sample size was also different for all polymers. Choosing polymer composition and LA conformation it is possible to choose desired release profile for the tested drug. Copyright © 2008 John Wiley & Sons, Ltd.

An effective route for the synthesis of aliphatic polyesters made from adipic or sebacic acid and alkanediols, using inorganic acid as a catalyst is reported. The monomer composition, reaction time, catalyst type, and reaction conditions were optimized to yield polyesters with weight average molecular weights of 23,000 for adipic acid and 85,000 for sebacic acid-based polyesters. The polymers melt at temperatures of 52–65°C and possess melt viscosity in the range of 5600–19,400cP. This route represents an alternative method for producing aliphatic polyesters for possible use in the preparation of degradable disposable medical supplies.

Magnetic nanoparticles (MNPs) are a new and promising addition to the spectrum of biomedicines. Their promise revolves around the broad versatility and biocompatibility of the MNPs and their unique physicochemical properties. Guided by applied external magnetic fields, MNPs represent a cutting-edge tool designed to improve diagnosis and therapy of a broad range of inflammatory, infectious, genetic and degenerative diseases. Magnetic hyperthermia, targeted drug and gene delivery, cell tracking, protein bioseparation and tissue engineering are but a few applications being developed for MNPs. MNPs toxicities linked to shape, size and surface chemistry are real and must be addressed before clinical use is realized. This article presents both the promise and perils of this new nanotechnology, with an eye towards opportunity in translational medical science.

Magnetic nanoparticles (MNPs) accumulate at disease sites with the aid of magnetic fields; biodegradable MNPs can be designed to facilitate drug delivery, influence disease diagnostics, facilitate tissue regeneration and permit protein purification. Because of their limited toxicity, MNPs are widely used in theranostics, simultaneously facilitating diagnostics and therapeutics. To realize therapeutic end points, iron oxide nanoparticle cores (5–30 nm) are encapsulated in a biocompatible polymer shell with drug cargos. Although limited, the toxic potential of MNPs parallels magnetite composition, along with shape, size and surface chemistry. Clearance is hastened by the reticuloendothelial system. To surmount translational barriers, the crystal structure, particle surface and magnetic properties of MNPs need to be optimized. With this in mind, we provide a comprehensive evaluation of advancements in MNP synthesis, functionalization and design, with an eye towards bench-to-bedside translation.

We propose a method for obtaining superparamagnetic nanoparticles based on iron oxide and their water suspensions. The structure and size of nanoparticles were confirmed by transmission electron microscopy, dynamic light scattering, and X-ray diffraction analysis. The nanoparticles also contained a fluorescent dye Dil C18. Cytotoxicity of obtained aqueous suspension was studied by MTT assay; low toxicity of nanoparticles was demonstrated. High T2-relaxivity of nanoparticles allows using them as a contrast agent for MRI. After incubation of cerebellar sections with nanoparticles vectorized with antibodies to antigen AMVB1, specific visualization of blood vessels was detected.

Background: Macrophage-carried nanoformulated catalase (‘nanozyme’) attenuates neuroinflammation and protects nigrostriatal neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. This is facilitated by effective enzyme transfer from blood-borne macrophages to adjacent endothelial cells and neurons leading to the decomposition of reactive oxygen species. Materials & methods: We examined the intra- and inter-cellular trafficking mechanisms of nanozymes by confocal microscopy. Improved neuronal survival mediated by nanozyme-loaded macrophages was demonstrated by fluorescence-activated cell sorting. Results: In macrophages, nanozymes were internalized mainly by clathrin-mediated endocytosis then trafficked to recycling endosomes. The enzyme is subsequently released in exosomes facilitated by bridging conduits. Nanozyme transfer from macrophages to adjacent cells by endocytosis-independent mechanisms diffusing broadly throughout the recipient cells. In contrast, macrophage-free nanozymes were localized in lysosomes following endocytic entry. Conclusion: Facilitated transfer of nanozyme from cell to cell can improve neuroprotection against oxidative stress commonly seen during neurodegenerative disease processes. Original submitted 29 June 2011; Revised submitted 14 September 2011; Published online 11 January 2012

To reduce formulation viscosity of bupivacaine/poly(DL lactic acid co castor oil) 3:7 without increasing bupivacaine release rates. Poly(DL lactic acid) 3:7 was synthesized and bupivacaine formulation prepared by mixing with additives ricinoleic acid or castor oil. In vitro release measurements identified optimum formulation. Anesthetized ICR mice were injected around left sciatic nerve using nerve stimulator with 0.1 mL of formulation. Animals received 10% bupivacaine-polymer formulation with 10% castor oil (p(DLLA:CO)3:7–10% bupi-10% CO) or 15% bupivacaine-polymer with 10% castor oil (p(DLLA:CO)3:7–15% bupi-10% CO). Sensory and motor block were measured. Viscosity of 10% and 15% bupivacaine-p(DLLA:CO)3:7 formulations was reduced using hydrophobic additives; however, castor oil reduced bupivacaine release rates and eliminated burst effect. Less than 10% of the incorporated bupivacaine was released during 6 h, and less than 25% released in 24 h in vitro. In vivo formulation injection resulted in a 24 h motor block and a sensory block lasting at least 72 h. Incorporation of hydrophobic low-viscosity additive reduced viscosity in addition to burst release effects. Bupivacaine-polymer formulation with castor oil additive demonstrated prolonged sensory analgesia in vivo, with reduced duration of motor block.

Pain following surgery is often treated by local anesthetic agents. Duration of the analgesia can be extended safely following administration of encapsulated large doses of local anesthetic agents.This review considers formulations used for encapsulation of local anesthetic agents for prolonged anesthesia effect. All studies describing encapsulation of a commercial local anesthetic agent for providing prolonged analgesia were considered using the NCBI Medline site. of local anesthetic, prolonged anesthesia, polymers and liposomes were entered in order to retrieve appropriate articles and reviews from 1966 to 2010, with emphasis on the last 10 years. Reference pages were searched manually for other relevant articles. The topics covered include an overview of local anesthetic agents and a review of local anesthetic carrier agents, with emphasis on liposomes and polymer carriers. Articles were limited to the English language.The current research areas for prolongation of local anesthetic effect are evaluated, along with their limitations. Each topic has been summarized, and the review has attempted to cover all current laboratory and clinical studies in a simple manner that should also be useful for readers without a pharmacology background. The direction of research is promising and exciting, and this review should be a useful up-to-date reference.Many formulations including polymer and liposome carriers have facilitated prolonged local anesthetic action for several days, although few clinical studies have been performed. This field promises a safe way to deliver local anesthetics for effect far beyond that of commercially available agents, with potential cost and health benefits for patients suffering chronic or postoperative pain.

To evaluate a new formulation of bupivacaine loaded in an injectable fatty acid based biodegradable polymer poly(lactic acid co castor oil) in prolonging motor and sensory block when injected locally.The polyesters were synthesized from DL: -lactic acid and castor oil with feed ratio of 4:6 and 3:7 w/w. Bupivacaine was dispersed in poly(fatty ester) liquid and tested for drug release in vitro. The polymer p(DLLA:CO) 3:7 loaded with 10% bupivacaine was injected through a 22G needle close to the sciatic nerve of ICR mice and the duration of sensory and motor nerve blockade was measured.The DL: -lactic acid co castor oil p(DLLA:CO) 3:7 released 65% of the incorporated bupivacaine during 1 week in vitro. Single injection of 10% bupivacaine loaded into this polymer caused motor block that lasted 24 h and sensory block that lasted 48 h.Previously we developed a ricinoleic acid based polymer with incorporated bupivacaine which prolonged anesthesia to 30 h. The new polymer poly(lactic acid co castor oil) 3:7 provides slow release of effective doses of the incorporated local anesthetic agent and prolongs anesthesia to 48 h.