RESUMEN
In the current study, core-crosslinked polymeric micelles (DEX-PMs) loaded with three different DEX derivatives designed to display different drug release kinetics, were evaluated for cancer therapy and compared to another effective nanomedicine formulation (long-circulating liposomes encapsulating dexamethasone, LCL-DEX). Pharmacokinetic studies with both radiolabeled dexamethasone and polymer showed that these polymeric systems have long circulating half-lives and may accumulate at the tumor site to a higher extent than liposomes. The in vitro drug release profiles and circulating drug levels in the blood stream show that DEX-PMs with dexamethasone covalently entrapped via a sulfone ester-containing linker (DMSL2) have prolonged circulation time and intermediate drug release kinetics compared to the other polymeric DEX-releasing systems. Furthermore, as the free dexamethasone circulating levels were similar when administered as DMSL2-PM or LCL-DEX, these systems were evaluated simultaneously for antitumor efficacy in B16F10 melanoma bearing mice. The corticosteroid-targeted systems inhibited tumor growth to a similar extent and both increased survival compared to free drug. Recently antitumor efficacy of targeted formulations has been correlated with a systemic effect: a decrease of white blood cell count. In this study all three polymeric systems, liposomes as well as free drug had similar effects on the number of circulating white blood cells, although white blood cell counts recovered faster in the group receiving free drug. In conclusion, corticosteroid-targeting with a polymeric system or a liposomal system translates in similar therapeutic effects. The proven high versatility of the PM with possible optimization and adjustment of the drug release to that required by the therapeutic application, clearly demonstrates the potential of these systems for the treatment of chronic inflammatory diseases including cancer.
Asunto(s)
Antineoplásicos/administración & dosificación , Dexametasona/administración & dosificación , Melanoma Experimental/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Dexametasona/química , Dexametasona/farmacocinética , Liposomas , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Micelas , Polímeros/administración & dosificación , Polímeros/química , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Distribución Tisular , Carga Tumoral/efectos de los fármacosRESUMEN
In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS-SEM revealed that early in vitro-formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS-SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.
Asunto(s)
Matriz Ósea/química , Matriz Ósea/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Espectrometría Raman/métodos , Animales , Células Cultivadas , Masculino , Osteocitos/metabolismo , Osteocitos/ultraestructura , RatasRESUMEN
Carbonated hydroxyapatite (CHA) coatings were applied onto titanium implants by using a biomimetic precipitation method. Different antibiotics were incorporated into the CHA coatings and their release and efficacy against bacteria growth were studied in vitro. The following antibiotics were used within this study: cephalothin, carbenicillin, amoxicillin, cefamandol, tobramycin, gentamicin and vancomycin. Increased concentrations of antibiotics in the coating solution led to a higher quantity of antibiotic incorporated into the CHA coating. Some antibiotics were better incorporated than others depending on their chemical structure. Antibiotics, containing carboxylic groups such as cephalothin, carbenicillin and cefamandol, were better incorporated than antibiotics lacking these groups. A bacterial inhibition test on Staphylococcus aureus bacteria showed inhibition of growth for all antibiotics that were released from the CHA coating. A release test was conducted in phosphate buffer saline PBS at pH 7.4 and 37 degrees C and showed that antibiotics containing carboxylic groups like cephalothin were slower released from the CHA coating than others. These results suggest that certain antibiotics are able to bind/chelate with calcium, resulting in a better incorporation into the CHA coating and a slower release. Antibiotics incorporated in CHA coatings on titanium implants might be used to prevent post-surgical infections and to promote bone-bonding of orthopedic devices.
Asunto(s)
Antibacterianos/química , Materiales Biocompatibles Revestidos/química , Durapatita/química , Titanio/química , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Materiales Biocompatibles Revestidos/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Staphylococcus aureus/efectos de los fármacosRESUMEN
Biomimetically deposited octacalcium phosphate (OCP) and carbonate apatite (BCA) as well as electrolytically deposited carbonate apatite (ECA) were considered as promising alternatives to conventional plasma spraying hydroxyapatite. This study compared their physicochemical characteristics and cell attachment behavior. The physicochemical characteristics included scanning electron microscopy observation, X-ray diffraction analysis, Fourier transform infrared spectroscopy analysis, surface roughness, coating thickness, dissolution test and scratch test. Cell attachment tests included morphology observation with stereomicroscopy and scanning electron microscopy as well as cell number count with DNA content assay. The OCP coating had 100% crystallinity and was about 40 microm thick, composed of large plate-like crystals of 30 microm, with the lowest surface roughness (R(a)=2.33 microm). The BCA coating had 60% crystallinity and was approximately 30 microm in thickness, composed of small crystals of 1-2 microm in size, with the highest surface roughness (R(a)=4.83 microm). The ECA coating had intermediate characteristics, with 78% crystallinity, 45 microm thickness, crystals of 5-6 microm and an average roughness of 3.87 microm. All coatings could be seen by eyes dissolving quickly and completely into acidic simulated body fluid (simulated physiological solutions-SPS, pH 3.0) but slowly and incompletely into neutral SPS (pH 7.3). It was suggested that the main factor determining coating dissolution in acidic SPS was the solubility isotherm, while some other factors including crystallinity and crystal size joined to determine coating dissolution in neutral SPS. In regard to adhesive strength, results of scratch test showed the critical load at the first crack of coating (L(c1)) was tightly related to crystal size as well as their arrangement, while the critical load at the total delamination of coating (L(c2)) was also related to the coating thickness. The ECA coating had the highest values. Owing to higher dissolution rate and globular appearance, BCA coating demonstrated the best goat bone marrow stromal cells attachment at 1 day or 3 days, followed by OCP and ECA coating.
Asunto(s)
Materiales Biomiméticos/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Fosfatos de Calcio/química , Materiales Biocompatibles Revestidos/química , Cristalización/métodos , Ensayo de Materiales/métodos , Titanio/química , Adsorción , Aleaciones , Animales , Materiales Biomiméticos/síntesis química , Adhesión Celular/fisiología , Células Cultivadas , Materiales Biocompatibles Revestidos/síntesis química , Electrólisis/métodos , Cabras , Dureza , Conformación Molecular , Propiedades de SuperficieRESUMEN
Calcium phosphate coatings containing an antibiotic were produced on titanium alloy (Ti6Al4V) implants using a biomimetic approach. Thin, amorphous calcium phosphate (ACP) coatings were first deposited onto Ti6Al4V plates by immersion in 5 times concentrated simulated body fluid (SBF), for 24h at 37 degrees C. The ACP-coated implants were then immersed in a supersaturated calcium phosphate (SCP) solution containing 0, 100, 200, 400, 600 or 800 mg/l of tobramycin for 48 h at 37 degrees C. A carbonated hydroxyapatite (CHA) layer, approximately 40 microm thick, was formed. Approximately 3 microg/mg of tobramycin was co-precipitated with the CHA crystals onto titanium alloy plates, using 800mg/l tobramycin in the coating solution. For comparison, plasma-sprayed calcium phosphate coatings were also immersed in solutions containing 100, 200, 400 or 1,000 mg/l of tobramycin for 10, 40 min, or 48 h. A maximum of about 0.3 microg/mg could be adsorbed onto the plasma-sprayed calcium phosphate coating with the comparable concentration of 800 mg/l in solution. The dissolution of coating and release of tobramycin were also measured in vitro using saline solution buffered at pH 5.0 or 7.3 at 37 degrees C. The release rate of tobramycin was faster at pH 7.3 than at pH 5, with 50 and 4 microg/ml/min, respectively. Tobramycin released from the biomimetic-coated plates could inhibit growth of Staphylococcus aureus bacteria. The result of this study, therefore, indicates that the biomimetic CHA coatings containing antibiotics could be used to prevent post-surgical infections in orthopaedic or trauma.
Asunto(s)
Antibacterianos/química , Durapatita/química , Imitación Molecular , Titanio/química , Tobramicina/química , Antibacterianos/farmacología , Fosfatos de Calcio , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Tobramicina/farmacología , Difracción de Rayos XRESUMEN
The use of monoclonal antibodies (MAbs) directed against tumor-associated antigens for targeting of photosensitizers is an interesting option to improve the selectivity of photodynamic therapy (PDT). Hydrophilic photosensitizers are most suitable for conjugation to MAbs because of their water solubility. The photosensitizer aluminum (III) phthalocyanine tetrasulfonate [AlPc(SO3H)4] has many ideal photochemical properties; however, because of its hydrophilicity, the free form of this sensitizer does not readily reach the critical intracellular target and, therefore, is ineffective in PDT. On the basis of our previous studies, we hypothesized that AlPc(SO3H)4 might be suitable for PDT when coupled to internalizing tumor-selective MAbs. In this study, a reproducible procedure is presented for coupling of AlPc(SO3H)4 to MAbs via the tetra-glycine derivative AlPc(SO2Ngly)4. Conjugation was performed to chimeric MAb (cMAb) U36 and murine MAbs (mMAb) E48 and 425 using a labile ester. Conjugates showed preservation of integrity and immunoreactivity and full stability in serum in vitro. At molar ratios >4, the solubility of the conjugates decreased. Data on the in vitro efficacy of PDT showed that in the chosen experimental setup the internalizing AlPc(SO2Ngly)4-mMAb 425 conjugate was about 7500 times more toxic to A431 cells than the free sensitizer (IC50s, 0.12 nM versus 900 nM). The AlPc(SO2Ngly)4-mMAb 425 conjugate was also more toxic than meta-tetrahydroxyphenylchlorin-mMAb 425 conjugates and free meta-tetrahydroxyphenylchlorin that had been tested previously (M. B. Vrouenraets et al., Cancer Res., 59: 1505-1513, 1999) in the same system (IC50s, 7.3 nm and 2.0 nM, respectively). Biodistribution analysis of AlPc(SO2Ngly)4-125I-labeled cMAb U36 conjugates with different sensitizer:MAb ratios in squamous cell carcinoma-bearing nude mice revealed selective accumulation in the tumor, although to a lesser extent than for the unconjugated 125I-labeled cMAb U36, whereas tumor:blood ratios were similar. These findings indicate that AlPc(SO3H)4 has high potential for use in PDT when coupled to internalizing tumor-selective MAbs.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoconjugados/química , Indoles/química , Compuestos Organometálicos/química , Fotoquimioterapia/métodos , Fármacos Sensibilizantes a Radiaciones/química , Animales , Anticuerpos Monoclonales/farmacocinética , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Inmunoconjugados/farmacocinética , Inmunoterapia/métodos , Indoles/administración & dosificación , Indoles/farmacocinética , Ratones , Ratones Desnudos , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/farmacocinética , Control de Calidad , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Reproducibilidad de los Resultados , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Coupling of photosensitizers to tumor-selective monoclonal antibodies (MAbs) is an attractive option for improving the selectivity of photodynamic therapy (PDT). For this purpose, hydrophilic sensitizers would be most suitable because of their solubility in water. However, such sensitizers are known to be ineffective in PDT, probably because they cannot readily pass the cell membrane and reach the critical intracellular target. We used the model compound TrisMPyP-PhiCO(2)H, a hydrophilic porphyrin derivative, to test the hypothesis that hydrophilic photosensitizers might become of therapeutic value when directed into the tumor cell by use of internalizing MAbs. TrisMPyP-PhiCO(2)H was conjugated using a labile ester. Conjugates showed no impairment of integrity on SDS-PAGE, full stability in serum in vitro, and optimal immunoreactivity when the sensitizer:MAb ratio was =3. At higher molar ratios, the solubility of the conjugates decreased. In vitro internalization experiments showed that TrisMPyP-PhiCONH-(125)I-cMAb U36 and TrisMPyPPhiCONH-(125)I-mMAb 425 conjugates were internalized by A431 cells, in contrast to TrisMPyP-PhiCONH-(125)I-mMAb E48 conjugates. Data on the in vitro efficacy of PDT with MAb-conjugated TrisMPyP-PhiCO(2)H showed that the internalizing cMAb U36 and mMAb 425 conjugates were phototoxic to A431 cells, while the non-internalizing E48 conjugate and the unconjugated sensitizer were not. Biodistribution data of conjugates with sensitizer:(125)I-cMAb U36 ratios varying from 1:1 to 3:1 in tumor-bearing nude mice revealed selective accumulation in the tumor. Conjugates with higher molar ratios were cleared more rapidly from the blood than the unconjugated (125)I-cMAb U36, resulting in lower tumor uptake but similar tumor-to-blood ratios. Our data suggest that hydrophilic photosensitizers might have therapeutic value when targeted to tumors by internalizing MAbs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Inmunoconjugados/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Radioisótopos de Yodo , Marcaje Isotópico/métodos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacocinética , Porfirinas/química , Porfirinas/farmacocinética , Distribución Tisular , Células Tumorales CultivadasRESUMEN
A limitation of photodynamic therapy is the lack of tumor selectivity of the photosensitizer. To overcome this problem, a protocol was developed for coupling of meta-tetrahydroxyphenylchlorin (mTHPC), one of the most promising photosensitizers, to tumor-selective monoclonal antibodies (MAbs). mTHPC was radiolabeled with 131I to facilitate the assessment of the in vitro and in vivo behavior. After the modification to 131I-mTHPC-(CH2COOH)4, thus increasing the water solubility and creating a functional moiety suitable for coupling, conjugation was performed using a labile ester. Insoluble aggregates were not formed when mTHPC-MAb conjugates with a molar ratio of up to 4 were prepared. These conjugates showed a minimal impairment of the integrity on SDS-PAGE, full stability in serum in vitro, and an optimal immunoreactivity. To test the in vivo behavior of the mTHPC-MAb conjugates, the head and neck squamous cell carcinoma-selective chimeric MAb U36 was used in head and neck squamous cell carcinoma-bearing nude mice. Biodistribution data showed that the tumor selectivity of cMAb U36-conjugated mTHPC was increased in comparison with free mTHPC, despite the fact that conjugates with a higher mTHPC:MAb ratio were more rapidly cleared from the blood. Preliminary results on the in vitro efficacy of photodynamic therapy with MAb-conjugated mTHPC showed that mTHPC coupled to the internalizing murine MAb 425 exhibited more phototoxicity than when coupled to the noninternalizing chimeric MAb U36.