RESUMO
Decades of research into the topic of oral nanoparticle (NP) delivery has still not provided a clear consensus regarding which properties produce an effective oral drug delivery system. The surface properties-charge and bioadhesiveness-as well as in vitro and in vivo correlation seem to generate the greatest number of disagreements within the field. Herein, a mechanism underlying the in vivo behavior of NPs is proposed, which bridges the gaps between these disagreements. The mechanism relies on the idea of biocoating-the coating of NPs with mucus-which alters their surface properties, and ultimately their systemic uptake. Utilizing this mechanism, several coated NPs are tested in vitro, ex vivo, and in vivo, and biocoating is found to affect NPs size, zeta-potential, mucosal diffusion coefficient, the extent of aggregation, and in vivo/in vitro/ex vivo correlation. Based on these results, low molecular weight polylactic acid exhibits a 21-fold increase in mucosal diffusion coefficient after precoating as compared to uncoated particles, as well as 20% less aggregation, and about 30% uptake to the blood in vivo. These discoveries suggest that biocoating reduces negative NP charge which results in an enhanced mucosal diffusion rate, increased gastrointestinal retention time, and high systemic uptake.
Assuntos
Portadores de Fármacos , Nanopartículas , Administração Oral , Sistemas de Liberação de Medicamentos/métodos , Muco , PolímerosRESUMO
Polymeric microspheres (MSs) have received attention for their potential to improve the delivery of drugs with poor oral bioavailability. Although MSs can be absorbed into the absorptive epithelium of the small intestine, little is known about the physiologic mechanisms that are responsible for their cellular trafficking. In these experiments, nonbiodegradable polystyrene MSs (diameter range: 500 nm to 5 µm) were delivered locally to the jejunum or ileum or by oral administration to young male rats. Following administration, MSs were taken up rapidly (≤ 5 min) by the small intestine and were detected by transmission electron microscopy and confocal laser scanning microscopy. Gel permeation chromatography confirmed that polymer was present in all tissue samples, including the brain. These results confirm that MSs (diameter range: 500 nm to 5 µm) were absorbed by the small intestine and distributed throughout the rat. After delivering MSs to the jejunum or ileum, high concentrations of polystyrene were detected in the liver, kidneys, and lungs. The pharmacologic inhibitors chlorpromazine, phorbol 12-myristate 13-acetate, and cytochalasin D caused a reduction in the total number of MSs absorbed in the jejunum and ileum, demonstrating that nonphagocytic processes (including endocytosis) direct the uptake of MSs in the small intestine. These results challenge the convention that phagocytic cells such as the microfold cells solely facilitate MS absorption in the small intestine.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Trânsito Gastrointestinal/fisiologia , Absorção Intestinal/fisiologia , Intestino Delgado/metabolismo , Microesferas , Poliestirenos/farmacocinética , Animais , Clorpromazina/farmacologia , Cromatografia em Gel , Citocalasina D/farmacologia , Absorção Intestinal/efeitos dos fármacos , Intestino Delgado/ultraestrutura , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Poliestirenos/administração & dosagem , Ratos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Numerous therapeutics demonstrate optimal absorption or activity at specific sites in the gastrointestinal (GI) tract. Yet, safe, effective pill retention within a desired region of the GI remains an elusive goal. We report a safe, effective method for localizing magnetic pills. To ensure safety and efficacy, we monitor and regulate attractive forces between a magnetic pill and an external magnet, while visualizing internal dose motion in real time using biplanar videofluoroscopy. Real-time monitoring yields direct visual confirmation of localization completely noninvasively, providing a platform for investigating the therapeutic benefits imparted by localized oral delivery of new and existing drugs. Additionally, we report the in vitro measurements and calculations that enabled prediction of successful magnetic localization in the rat small intestines for 12 h. The designed system for predicting and achieving successful magnetic localization can readily be applied to any area of the GI tract within any species, including humans. The described system represents a significant step forward in the ability to localize magnetic pills safely and effectively anywhere within the GI tract. What our magnetic pill localization strategy adds to the state of the art, if used as an oral drug delivery system, is the ability to monitor the force exerted by the pill on the tissue and to locate the magnetic pill within the test subject all in real time. This advance ensures both safety and efficacy of magnetic localization during the potential oral administration of any magnetic pill-based delivery system.
Assuntos
Absorção Intestinal/fisiologia , Intestino Delgado/diagnóstico por imagem , Intestino Delgado/fisiologia , Magnetismo , Farmacocinética , Animais , Fluoroscopia , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Although other methods exist for monitoring gastrointestinal motility and contractility, this study exclusively provides direct and quantitative measurements of the forces experienced by an orally ingested pill. We report motive forces and torques calculated from real-time, in vivo measurements of the movement of a magnetic pill in the stomachs of fasted and fed humans. Three-dimensional net force and two-dimensional net torque vectors as a function of time data during gastric residence are evaluated using instantaneous translational and rotational position data. Additionally, the net force calculations described can be applied to high-resolution pill tracking acquired by any modality. The fraction of time pills experience ranges of forces and torques are analyzed and correlate with the physiological phases of gastric digestion. We also report the maximum forces and torques experienced in vivo by pills as a quantitative measure of the amount of force pills experience during the muscular contractions leading to gastric emptying. Results calculated from human data are compared with small and large animal models with a translational research focus. The reported magnitude and direction of gastric forces experienced by pills in healthy stomachs serves as a baseline for comparison with pathophysiological states. Of clinical significance, the directionality associated with force vector data may be useful in determining the muscle groups associated with gastrointestinal dysmotility. Additionally, the quantitative comparison between human and animal models improves insight into comparative gastric contractility that will aid rational pill design and provide a quantitative framework for interpreting gastroretentive oral formulation test results.
Assuntos
Estômago/fisiologia , Torque , Animais , Cães , Ingestão de Alimentos , Eletromiografia , Jejum , Humanos , Imageamento Tridimensional , RatosRESUMO
Systemic lupus erythematosus development is influenced by both sex and the gut microbiota. Metabolite production is a major mechanism by which the gut microbiota influences the immune system, and we have previously found differences in the fecal metabolomic profiles of lupus-prone female and lupus-resistant male BWF1 mice. Here we determine how sex and microbiota metabolite production may interact to affect lupus. Transcriptomic analysis of female and male splenocytes showed genes that promote phagocytosis were upregulated in BWF1 male mice. Because patients with systemic lupus erythematosus exhibit defects in macrophage-mediated phagocytosis of apoptotic cells (efferocytosis), we compared splenic macrophage efferocytosis in vitro between female and male BWF1 mice. Macrophage efferocytosis was deficient in female compared to male BWF1 mice but could be restored by feeding male microbiota. Further transcriptomic analysis of the genes upregulated in male BWF1 mice revealed enrichment of genes stimulated by PPARγ and LXR signaling. Our previous fecal metabolomics analyses identified metabolites in male BWF1 mice that can activate PPARγ and LXR signaling and identified one in particular, phytanic acid, that is a very potent agonist. We show here that treatment of female BWF1 splenic macrophages with phytanic acid restores efferocytic activity via activation of the PPARγ and LXR signaling pathways. Furthermore, we found phytanic acid may restore female BWF1 macrophage efferocytosis through upregulation of the proefferocytic gene CD36. Taken together, our data indicate that metabolites produced by BWF1 male microbiota can enhance macrophage efferocytosis and, through this mechanism, could potentially influence lupus progression.
Assuntos
Lúpus Eritematoso Sistêmico , Microbiota , Camundongos , Masculino , Feminino , Animais , PPAR gama , Ácido Fitânico , Camundongos Endogâmicos NZB , Macrófagos , Fagocitose , Transdução de SinaisRESUMO
We report on the development of a modified solvent removal method for the encapsulation of hydrophilic drugs within poly(lactic-co-glycolic acid) (PLGA). Using a water/oil/oil double emulsion, hydrophilic doxycycline was encapsulated within PLGA spheres with particle diameters ranging from approximately 600 nm to 19 µm. Encapsulation efficiencies of up to 74% were achieved for theoretical loadings from 1% to 10% (w/w), with biphasic release over 85 days with nearly complete release at the end of this time course. About 1% salt was added to the formulations to examine its effects on doxycycline release; salt modulated release only by increasing the magnitude of initial release without altering kinetics. Fourier transform infrared spectroscopy indicated no characteristic differences between doxycycline-loaded and control spheres. Differential scanning calorimetry and X-ray diffraction suggest that there may be a molecular dispersion of the doxycycline within the spheres and the doxycycline may be in an amorphous state, which could explain the slow, prolonged release of the drug.
Assuntos
Doxiciclina/administração & dosagem , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos , Antibacterianos/administração & dosagem , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Preparações de Ação Retardada , Emulsões , Interações Hidrofóbicas e Hidrofílicas , Ácido Láctico , Microscopia Eletrônica de Varredura , Microesferas , Nanosferas/química , Tamanho da Partícula , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solventes , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
BACKGROUND: All-trans retinoic acid (ATRA) is a biologically active isomer of retinoic acid (RA). Topical ATRA (retin-a, retin-a micro, atralin, renova, and avita) is the active pharmaceutical ingredient for FDA-approved treatments for acne and skin wrinkles. Oral formulations (Vesanoid) treat acute promyelocytic leukemia, but oral dosing can induce severe side effects. Despite benefits in various rodent models of inflammatory bowel disease (IBD), toxicity and controversial clinical observations have diminished enthusiasm for ATRA IBD clinical trials. To circumvent these issues and to use ATRA's key role in maintaining gut tolerance, we developed a poly(lactic-co-glycolic acid) (PLGA) microsphere (MS) encapsulated ATRA formulation aimed at directing ATRA delivery to immune structures of the gut, limiting systemic exposure. Initially, ATRA MS was developed as a component of a combinatorial product (TreXTAM) that also contained encapsulated transforming growth factor (TGF)-ß and ATRA in a 1:2 w/w ratio. Although the combination was optimal, benefit was also observed when ATRA MS was given alone in the CD4+ CD25-T-cell adoptive transfer (ACT) colitis model. METHODS: We used the ACT and DSS-induced murine models of colitis to expand on the dose-dependent effects of oral ATRA MS when given alone. The DSS model was also used to compare the efficacy of ATRA MS and soluble ATRA, while healthy animals were used to compare the pharmacokinetics of the two drugs. RESULTS: In both the ACT and DSS-induced murine models of colitis, ATRA MS was observed to be effective in ameliorating disease. ATRA MS was also observed to be more effective than soluble ATRA in these models and displayed more favorable pharmacokinetics. CONCLUSIONS: We suggest ATRA MS, as a standalone product, may attenuate IBD and perhaps limit fibrosis, while limiting systemic side effects.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Camundongos , Roedores/metabolismo , Fator de Crescimento Transformador beta , Tretinoína/metabolismoRESUMO
BACKGROUND: TreXTAM® is a combination of the key regulatory cytokine transforming growth factor beta (TGFß) and all trans retinoic acid (ATRA) microencapsulated for oral delivery to immune structures of the gut. It is in development as a novel treatment for inflammatory bowel disease (IBD). AIM: To measure TGFß levels in blood and tissue after oral administration of encapsulated TGFß. METHODS: Animals were orally administered encapsulated TGFß by gavage. Levels of drug substance in blood and in gut tissues at various times after administration were measured by ELISA. RESULTS: We made the surprising discovery that oral administration of TreXTAM dramatically (approximately 50%) and significantly (P = 0.025) reduced TGFß levels in colon, but not small intestine or mesenteric lymph nodes. Similarly, levels in rat serum after 25 d of thrice weekly dosing with either TreXTAM, or microencapsulated TGFß alone (denoted as TPX6001) were significantly (P < 0.01) reduced from baseline levels. When tested in the SCID mouse CD4+CD25- adoptive cell transfer (ACT) model of IBD, oral TPX6001 alone provided only a transient benefit in terms of reduced weight loss. CONCLUSION: These observations suggest a negative feedback mechanism in the gut whereby local delivery of TGFß results in reduced local and systemic levels of the active form of TGFß. Our findings suggest potential clinical implications for use of encapsulated TGFß, perhaps in the context of IBD and/or other instances of fibrosis and/or pathological TGFß signaling.
RESUMO
The time-dependent bioadhesive performance of various polymers was evaluated using a texture analyzer apparatus and freshly excised rat small intestinal tissue. A series of novel bioadhesive polymers were prepared by conjugating L-phenylalanine, L-tyrosine, and L-DOPA to either a low molecular weight poly (butadiene-maleic anhydride) or a high molecular weight poly (ethylene-maleic anhydride). Bioadhesive force was characterized as a function of time relative to polycarbophil, a slightly cross-linked poly (acrylic acid)-derivative, revealing different fracture strengths and tensile work for each of the six backbone-side chain conjugations that were studied. While polycarbophil showed a rapid and significant loss of bioadhesion over the testing period, the newly developed synthetic polymers were able to maintain their bioadhesive performance over the course of 91 min with the overall magnitude of bioadhesion corresponding to the hydrogen bonding potential of the associated side chains. These results highlight the potential of these polymers for use in the development of more effective bioadhesive oral drug delivery systems.
Assuntos
Adesivos/síntese química , Mucosa Intestinal/química , Intestino Delgado/química , Levodopa/química , Fenilalanina/química , Tirosina/química , Resinas Acrílicas/química , Adesividade , Adesivos/metabolismo , Animais , Butadienos/química , Ligação de Hidrogênio , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Levodopa/metabolismo , Anidridos Maleicos/química , Fenilalanina/metabolismo , Polimerização , Ratos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Tirosina/metabolismoRESUMO
Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(l,d-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3 +/- 58.0 microm) rather than smaller (11.15 +/- 11.08 microm) microspheres, generated pores on the order of 200 microm as compared to 20 microm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.
Assuntos
Microesferas , Soroalbumina Bovina/química , Engenharia Tecidual , Animais , Bovinos , Adesão Celular , Linhagem Celular , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Soroalbumina Bovina/ultraestruturaRESUMO
Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.
Assuntos
Fator de Crescimento Insulin-Like I/química , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Fator de Crescimento Transformador beta1/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Portadores de Fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Fator de Crescimento Insulin-Like I/farmacocinética , Fator de Crescimento Insulin-Like I/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Fator de Crescimento Transformador beta1/farmacocinética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The bioadhesive polymer, poly(fumaric-co-sebacic) anhydride, p(FASA), was used to fabricate small diameter insulin microspheres and evaluate their in vivo performance in a type 1 diabetic rat as well as a type 1 diabetic dog model. The process of phase inversion nanoencapsulation was used to fabricate p(FASA) microspheres containing insulin. Using laser diffraction spectrometry, 90% of the microspheres used in the fed double dose rat experiments were found to have a volumetric diameter of 5.9 microm or smaller. In comparison, 90% of the microspheres used in fed single dose rat experiments were found to have a volumetric diameter of 2.6 microm or smaller while the microspheres used in the diabetic dog experiments were found to have a volumetric diameter of 1.2 microm or smaller. Insulin microspheres administered to diabetic rats in the fed double dose experiment produced a relative bioavailability (RB) of 23.3% while insulin microspheres administered to diabetic rats in the fed single dose experiment produced a RB of 5.5+/-1.7%. Insulin microspheres administered to fasted diabetic dogs produced a RB of 5.5+/-3.4%.
Assuntos
Ácidos Decanoicos/química , Fumaratos/química , Insulina/uso terapêutico , Microesferas , Polímeros/química , Administração Oral , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Cães , Feminino , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Insulina/química , Insulina/farmacocinética , Masculino , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Ratos , Ratos Endogâmicos BBRESUMO
Stem and non-stem cell behavior is heavily influenced by the surrounding microenvironment, which includes other cells, matrix, and potentially biomaterials. Researchers have been successful in developing scaffolds and encapsulation techniques to provide stem cells with mechanical, topographical, and chemical cues to selectively direct them toward a desired differentiation pathway. However, most of these systems fail to present truly physiological replications of the in vivo microenvironments that stem cells are typically exposed to in tissues. Thus, cell mimicking microparticles (CMMPs) have been developed to more accurately recapitulate the properties of surrounding cells while still offering ways to tailor what stimuli are presented. This nascent field holds the promise of reducing, or even eliminating, the need for live cells in select, regenerative medicine therapies, and diagnostic applications. Recent, CMMP-based studies show great promise for the technology, yet only reproduce a small subset of cellular characteristics from among those possible: size, morphology, topography, mechanical properties, surface molecules, and tailored chemical release to name the most prominent. This Review summarizes the strengths, weaknesses, and ideal applications of micro/nanoparticle fabrication and customization methods relevant to cell mimicking and provides an outlook on the future of this technology. Moving forward, researchers should seek to combine multiple techniques to yield CMMPs that replicate as many cellular characteristics as possible, with an emphasis on those that most strongly influence the desired therapeutic effects. The level of flexibility in customizing CMMP properties allows them to substitute for cells in a variety of regenerative medicine, drug delivery, and diagnostic systems. Stem Cells Translational Medicine 2018;7:232-240.
Assuntos
Micropartículas Derivadas de Células/fisiologia , Células-Tronco/citologia , Diferenciação Celular/fisiologia , Microambiente Celular/fisiologia , Humanos , Medicina Regenerativa/métodosRESUMO
Substrate stiffness is known to alter cell behavior and drive stem cell differentiation, though most research in this area has been restricted to traditional, two-dimensional culture systems rather than more physiologically relevant, three-dimensional (3D) platforms. In this study, we utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to human adipose derived stem cells in 3D spheroid culture to examine changes in adipogenic differentiation response and mechanophenotype. After 21 days of adipogenic induction, spheroids containing CMMPs (composite spheroids) stiffened in accordance with CMMP elasticity such that spheroids containing the stiffest, ~ 10 kPa, CMMPs were over 27% stiffer than those incorporating the most compliant, ~ 0.25 kPa CMMPs. Adipogenically induced, cell-only spheroids were over 180% larger and 50% more compliant than matched controls. Interestingly, composite spheroids cultured without chemical induction factors dissociated when presented with CMMPs stiffer than ~ 1 kPa, while adipogenic induction factors mitigated this behavior. Gene expression for PPARG and FABP4 were upregulated more than 45-fold in adipogenically induced samples compared to controls but were unaffected by CMMP elasticity, attributed to insufficient cell-CMMP contacts throughout the composite spheroid. In summary, mechanically tuned CMMPs influenced whole-spheroid mechanophenotype and stability but minimally affected differentiation response.
Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica , Mecanotransdução Celular , Esferoides Celulares/metabolismo , Células-Tronco/metabolismo , Tecido Adiposo/citologia , Feminino , Humanos , Pessoa de Meia-Idade , Esferoides Celulares/citologia , Células-Tronco/citologiaRESUMO
A quick fabrication method for making double-walled (DW) polymeric nanospheres is presented. The process uses sequential precipitation of two polymers. By choosing an appropriate solvent and non-solvent polymer pair, and engineering two sequential phase inversions which induces first precipitation of the core polymer followed by precipitation of the shell polymer, DW nanospheres can be created instantaneously. A series of DW formulations were prepared with various core and shell polymers, then characterized using laser diffraction particle sizing, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry (DSC). Atomic force microscopy (AFM) imaging confirmed existence of a single core polymer coated with a second polymer. Insulin (3.3% loading) was used as a model drug to assess its release profile from core (PLGA) and shell (PBMAD) polymers and resulted with a tri-phase release profile in vitro for two months. Current approaches for producing DW nanoparticles (NPs) are limited by the complexity and time involved. Additional issues include aggregation and entrapment of multiple spheres and the undesired formation of heterogeneous coatings. Therefore, the technique presented here is advantageous because it can produce NPs with distinct, core-shell morphologies through a rapid, spontaneous, self-assembly process. This method not only produces DW NPs, but can also be used to encapsulate therapeutic drug. Furthermore, modification of this process to other core and shell polymers is feasible using the general guidelines provided in this paper.
Assuntos
Portadores de Fármacos/química , Insulina/farmacologia , Nanosferas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Butadienos/química , Preparações de Ação Retardada/química , Liberação Controlada de Fármacos , Elastômeros/química , Excipientes/química , Concentração de Íons de Hidrogênio , Anidridos Maleicos/química , Tamanho da Partícula , Solventes/química , Propriedades de Superfície , Fatores de TempoRESUMO
Neoadjuvant immunotherapy with the combination of intratumoral IL-12 and TNF-alpha encapsulated in poly-lactic acid microspheres (PLAM) generate a greater systemic immune response than either cytokine alone. We sought to examine the effector cells responsible for this synergy using the poorly immunogenic B16 melanoma and MCA205 sarcoma cell lines. Splenocytes from MCA205 bearing mice treated with IL-12 and TNF-alpha PLAM contained significantly more tumor-specific IFN-gamma secreting cells than IL-12 alone. Adoptive transfer of lymphocytes from mice treated by the combination mediated significant tumor regression in mice bearing established pulmonary metastases. In mice bearing bilateral tumors, treatment of the primary with IL-12 and TNF-alpha PLAM, resulted in suppression of contralateral tumor growth. Both the local and distant effects were absent in mice depleted of CD8+ T-cells. In B16 bearing mice with established pulmonary disease, only the combination of intratumoral IL-12 and TNF-alpha resulted in a significant reduction of lung nodules. Both the local and distant effects were eradicated in mice depleted of either CD8+ T-cells or NK cells. The local and sustained release of IL-12 and TNF-alpha using PLAM synergistically activate both a cytotoxic T-cell and NK cell response, although their impact varies with MHC class I expression.
Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-12/farmacologia , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Melanoma/imunologia , Melanoma/secundário , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Transplante de Neoplasias , Sarcoma/tratamento farmacológico , Sarcoma/imunologia , Sarcoma/secundário , Linfócitos T Citotóxicos/imunologiaRESUMO
The method of phase inversion nanoencapsulation (PIN) and microencapsulation was used to produce biodegradable poly(fumaric-co-sebacic anhydride) (p(FASA)) microspheres that contain insulin. Microspheres were characterized by SEM and a laser light scattering technique to determine particle size distribution. Insulin stability was determined by RP and SEC HPLC. Release rate studies were conducted and microspheres were administered subcutaneously (SQ) to type 1 diabetic rats to determine the bioactivity of insulin at three different dosages. Pharmacokinetic parameters for SQ experiments were measured using the trapezoidal rule by plotting average plasma insulin level (PIL) vs. time and determining peak concentration (CP), the time of peak concentration (TP), duration of PIL curve (D), and relative bioavailability (RB). When our insulin containing formulation was analyzed by HPLC, there was no evidence of high molecular weight transformation (HMWT) or deamidated products. In addition, we effectively altered the onset, peak, and duration of insulin action after SQ injection.
Assuntos
Anidridos/administração & dosagem , Ácidos Decanoicos/administração & dosagem , Diabetes Mellitus Tipo 1/tratamento farmacológico , Fumaratos/administração & dosagem , Insulina/administração & dosagem , Polímeros/administração & dosagem , Anidridos/química , Animais , Disponibilidade Biológica , Glicemia/análise , Cromatografia Líquida de Alta Pressão , Ácidos Decanoicos/química , Fumaratos/química , Injeções Subcutâneas , Insulina/sangue , Insulina/farmacocinética , Insulina/uso terapêutico , Microscopia Eletrônica de Varredura , Polímeros/química , RatosRESUMO
Tumor cells, injected s.c., were maintained until spontaneous metastases to the lungs were established in all of the mice. Mice were then treated with a single dose of cytokine-encapsulated biodegradable microspheres injected directly into primary s.c. tumors to achieve a local and sustained release of interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination of these cytokines to the tumor microenvironment. The s.c. tumors were surgically excised 6 days after microsphere injections, and the mice were monitored for recurrence of the primary tumor, survival, and progression of metastatic disease. Combined neoadjuvant treatment with IL-12 and GM-CSF microspheres was superior to all other treatments in reducing the recurrence of primary tumors, enhancing postoperative survival, and suppressing established metastatic disease. Long-term survival analysis demonstrated that intratumoral injection of IL-12 + GM-CSF-loaded microspheres resulted in the complete cure of disseminated disease in the majority of the animals. The addition of systemic low-dose IL-2 therapy to the treatment protocol resulted in the loss of the antitumor activity induced by IL-12 + GM-CSF treatment. In vivo lymphocyte subset depletions established that both T- and natural killer-cell subsets were required for the suppression of primary and metastatic tumors. Long-term, tumor-specific T-cell activity was demonstrated by immunohistochemical analysis of metastatic lesions, IFN-gamma enzyme-linked immunosorbent spot (ELISPOT) assays and tumor challenge studies. These results establish that neoadjuvant in situ tumor immunotherapy with IL-12 + GM-CSF microspheres induces both innate and adaptive antitumor immune responses resulting in the eradication of disseminated disease.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias do Colo/terapia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Imunoterapia/métodos , Interleucina-12/administração & dosagem , Neoplasias Pulmonares/terapia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/cirurgia , Relação Dose-Resposta Imunológica , Sinergismo Farmacológico , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interferon gama/biossíntese , Interferon gama/sangue , Interferon gama/imunologia , Interleucina-12/sangue , Interleucina-12/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/cirurgia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microesferas , Terapia Neoadjuvante , Transplante de Neoplasias , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Inverse emulsification was used to fabricate polyacrylamide (PAAm) microbeads with size and elastic properties similar to typical, mammalian cells. These biomimicking microbeads could be fluorescently stained and functionalized with a collagen type-I coating, post-polymerization, for tracking bead locations and promoting cell recognition/binding, respectively. By occupying a previously unfilled range of sizes and mechanical properties, these microbeads may find unique use in both biomedical and materials applications.
Assuntos
Resinas Acrílicas/síntese química , Microesferas , Células/citologia , Elasticidade , Emulsões , Tamanho da PartículaRESUMO
A model enzyme, carbonic anhydrase, was encapsulated and released from poly(lactide-co-glycolide) (PLGA) microspheres (1-3 microm) made by a novel phase inversion technique. Lecithin was used as a surfactant in the encapsulation process and was incorporated in either the organic phase, aqueous phase, both phases, or not at all. Additional microspheres were also made with lecithin incorporated in the aqueous phase and a basic salt, MgCO3, in the polymeric phase. Released carbonic anhydrase, protein extracted from microspheres, or enzyme incubated with lecithin and PLGA were analyzed via HPLC and activity assay to determine the effect of these additives on protein integrity and activity. Lecithin in the aqueous phase appeared to increase the fraction of enzyme in monomeric form as well as its activity for both extracted protein and released protein as compared to the other formulations without MgCO3. Incubation of enzyme with PLGA degradation products indicated that the acidic environment within the microspheres aids in the irreversible inactivation of the encapsulated protein. Addition of MgCO3 further increased the amount of monomer in both the extracted and released protein by decreasing the amount of acid-induced cleavage and noncovalent aggregation, but still greatly decreased the activity of the enzyme.