- Email: [email protected]
Novel paclitaxel nanoparticles: Development, in vitro anti-tumor activity in BT-549 cells and in vivo evaluation
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
e119
Conclusion The results obtained in this preliminary work highlighted that the new complex PPS:chitosan can be easily prepared. Further studies will be carried out in vaginal environment in order to evaluate the influence of natural enzyme in the complex dissolution and in drug release. Acknowledgements The authors wish to thank Bene pharmaChem for the free PPS raw material. References [1] R. Peeker, M. Fall, Int Urogynecol J 11 (2000) 23–32. [2] V.R. Anderson, C.M. Perry, Drugs 66 (2006) 821–835. [3] O. Felt, P. Buri, R. Gurny, Drug Delivery and Industrial Pharmacy 24 (1998) 979–993.
doi:10.1016/j.jconrel.2010.07.090
Novel paclitaxel nanoparticles: Development, in vitro anti-tumor activity in BT-549 cells and in vivo evaluation
Fig. 3. PPS release test.
Gopal V. Shavi⁎, A. Ranjith Kumar, A. Karthik, M. Naseer, G. Aravind, B.D. Praful, M. Sreenivasa Reddy, N. Udupa Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka-576104, India ⁎Corresponding author. E-mail: [email protected]. Abstract summary The aim is to develop paclitaxel PLGA nanoparticles (NPs), for intravenous administration, with improved therapeutic efficacy using single-emulsion technique. Encapsulation efficiency was higher at polymer-to-drug ratio of 20:1 with particle size <200 nm. In vitro drug release exhibited biphasic pattern with initial burst release followed by slow and continuous release for 15 days. In vitro antitumor activity against BT-549 cells showed complete inhibition after 168 h of incubation. AUC and t1/2 were found to be higher for NPs.
Fig. 4. Chitosan release test.
The behavior of PPS release is quite different depending from the medium used. In fact, independently from the PPS content, in phosphate buffer only about 15% of active is released after 96 h instead of more than 50% is released at pH 3. Differently, the amount of chitosan dissolved is quite the same for both medium tested (about 30% after 96 h).
Introduction Paclitaxel (Taxol®) is one of the best antineoplastic agents found from nature in the past decades. Like many other anticancer drugs, there are difficulties in its clinical administration due to its poor solubility. To enhance its solubility and allow parenteral administration, paclitaxel is currently available as 6 mg/ml solution in a vehicle composed of 1:1 blend of cremophor EL and ethanol, but this has been found to cause adverse side-effects such as hypersensitivity reactions, nephrotoxicity, neurotoxicity as well as effects on endothelial and vascular muscles causing vasodilation, labored breathing, lethargy and hypotension. Nanoparticles of biodegradable polymers can provide an ideal solution to the adjuvant problem and realize a controlled delivery by passive targeting of the drug with better efficacy and fewer side-effects. Moreover, nanoparticles can escape from the vasculature through the leaky endothelial tissue that surrounds the tumor and then accumulate in certain solid tumors by the so-called Enhanced Permeation and Retention (EPR) effect. A cremophor free formulation of paclitaxel nanoparticles, Abraxane®, has been approved by FDA for recurrent metastatic breast cancer. Polymeric nanoparticles demonstrated that a significant improvement in drug specificity of action, this effect being mainly attributed to changes in tissue distribution and pharmacokinetics. The objective of this study was to develop a polymeric drug delivery system for paclitaxel with PLGA 50:50 intended to be intravenously administered, capable of improving the therapeutic index of the drug and devoid of the adverse effects of cremophor EL. In this study, the paclitaxel was encapsulated within PLGA 50:50
e120
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
using poly vinyl alcohol (PVA) as emulsifier, to stabilize the dispersed oil droplets which create a sustained-release system over a period of hours to days with improved anti-tumoral efficacy. Experimental methods Nanoparticles were prepared by simple interfacial deposition technique with different ratio of PLGA 50:50 to drug, (5:1, 10:1, 15:1 and 20:1) were dissolved in a certain amount (0.5 ml) of dichloromethane. The resulting solution, (organic phase) was added slowly to the aqueous phase containing PVA solution (1.0% w/v), homogenized using high speed homogenizer with a speed of 15,000 rpm followed by sonication in ice water bath for 3 min at 10 kV using probe sonicator to form nanoparticles. The resulting emulsion was kept under stirring up to 12 h for complete evaporation of organic solvent, centrifuged at 15,000 rpm for 30 min for separation of nanoparticles, obtained as the pellet further dispersed in mannitol solution (5 mg/ml) as cryoprotectant and lyophilized for 48 h at −40 °C with a vacuum pressure of 50 mm Torr to obtain dry fluffy mass. Compatibility studies of drug and carrier system were carried out using Differential Scanning Calorimetry (DSC) analysis. The influence of different experimental parameters such as polymer to drug ratio, organic to aqueous phase ratio, homogenization speed and sonication time on the encapsulation efficiency of paclitaxel in the nanoparticles was evaluated. Nanoparticles were evaluated for percentage encapsulation efficiency (amount of drug encapsulated in the nanoparticles was analyzed by HPLC method), particle size, polydispersity index, zeta potential, surface morphology and in vitro drug release studies using vial method. The effect of pure drug and paclitaxel nanoparticles (NPs) on the viability of BT-549 cells (breast cancer cells) was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The cells were incubated with the pure drug and paclitaxel nanoparticles suspension at concentrations of 0.01, 0.1, 1 and 10 μg/ml, respectively. At specific intervals of 24, 48, 72 and 168 h % cell viability was measured by a microtiter plate reader. Accelerated stability studies at 25 ± 2 °C and 60% ± 5% RH were carried out for the optimized formulation (20:1) for a period of 180 days. Pharmacokinetics of the pure drug and paclitaxel nanoparticles were carried out in Wistar rats (5 mg/kg) and the serum samples were analyzed using HPLC. Pharmacokinetic parameters i.e. AUC, elimination rate constant (ke), t1/2 and clearance (Cl) were determined and data obtained was statistically analyzed by using independent sample t-test with SPSS software 11.5 version, to stumble out significant differences between two groups. Statistical significance was established at p < 0.05.
Paclitaxel-loaded biodegradable drug delivery systems manufactured from single emulsion technique with PLGA 50:50 were reproducible with highest encapsulation efficiency (94.5 ± 4.2) for the polymer to drug ratio of 20:1. The particle size, polydispersity index and zeta potential were found to be in the range of 150–200 nm, 0.257– 0.425 and −33.5 to −68.9 respectively. The preparation allowed the formation of spherical nanometric, homogeneous and negatively charged zeta potential, proved no aggregation and sedimentation with good polydispersity index. Surface morphology studies revealed smooth and spherical nature of the particles as shown in Fig. 2.
Fig. 2. Surface morphology of the polymeric nanoparticles.
The release behavior of paclitaxel from the developed nanoparticles exhibited a biphasic patter characterized by an initial fast release during the first 24 h, followed by a slower and continuous at extremely slow rates for a period of 15 days. The results of cell viability are shown in Fig. 3a and b, after 24 h of incubation time, no cytotoxic effect could be observed for any of the concentrations tested. However for each of the concentrations there was an enhancement in cytoxicity with increasing time of incubation. Cell growth was completely inhibited for paclitaxel nanoparticles after 168 h of incubation when compared to pure drug. Results of the stability studies revealed that the prepared nanoparticles are stable at 25 ± 2 °C and 60% ± 5% RH for a period of 180 days. The results of pharmacokinetic parameters are shown in Table 1. The values area under the concentration-versus-time curve (AUC) and t1/2 of paclitaxel NPs were found to be much higher (3–4 times) for paclitaxel vectored by PLGA (50:50) nanoparticles than for pure drug. Nanoparticles were relatively long circulating (t1/2 33.19 h) with low clearance rate when compared to pure drug.
Results and discussion The selection of an optimal formulation in the study was based on that which provided a combination of better morphology (in terms of sphericity and discreteness), extreme unaffected particle sizes and drug entrapment. DSC analysis indicated the absence of interaction between the drug and selected polymer and excipient, the same was shown in Fig.1.
Fig. 1. DSC analysis of a) paclitaxel and b) polymeric nanoparticles.
Fig. 3. a. Cell viability of BT-549 cells incubated with pure drug. b. Cell viability of BT549 cells incubated with paclitaxel NPs.
Abstracts / Journal of Controlled Release 148 (2010) e112–e124 Table 1 Pharmacokinetic parameters of pure drug and paclitaxel NPs after i.v. administration into rats. Values Parameter
Unit
Paclitaxel NPs
Pure drug (Taxol®)
AUC Elimination rate constant(Ke) t1/2 Clearance(Cl)
h μg/ml 1/h h L/h kg
19.032 0.0208 33.191 0.748
6.108 0.163 5.003 1.273
e121
these micelles (when core-crosslinked) display prolonged circulation and enhanced passive tumor accumulation due to the EPR effect, the loaded drug is rapidly diffused out after injection in the blood stream. So in this study doxorubicin, a cytotoxic drug, was selected for covalent attachment to the core of these micelles, using a methacrylated derivative of doxorubicin (Fig. 1b).
Conclusion The present study showed controlled delivery of paclitaxel from nanoparticles with enhanced anti-tumoral efficacy. Hence Paclitaxel NPs may be considered as an effective anticancer drug delivery system for long term treatment of breast cancer. Acknowledgements The authors are grateful to Getwell Pharmaceuticals PVT Ltd., Gurgaon, India for providing gift sample of paclitaxel. This study was also supported by Department of Science and Technology (DST), New Delhi, India. References [1] F. Danhier, N. Lecouturier, B. Vroman, C. Jérôme, J. Marchand-Brynaert, O. Feron, V. Préat, Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation, J. Control. Release 133 (2009) 11–17. [2] C. Fonseca, S. Simoes, R. Gaspar, Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity, J. Control. Release 133 (83) (2002) 273–286.
doi:10.1016/j.jconrel.2010.07.091
Fig. 1. (a) Chemical structure of micelle forming methacrylated mPEG-b-pHPMAm-Lacn, (b) methacrylated derivative of doxorubicin with a hydrolyzable hydrazone linker.
This prodrug could be co-crosslinked in the hydrophobic core of the micelles composed of methacrylated block copolymers (Fig. 1a) by means of KPS and TEMED polymerization. Importantly, this doxorubicin derivative contains a biodegradable hydrazone linker, which is stable at physiological pH, but hydrolyzes in acidic environment [5]. Thus it was hypothesized that doxorubicin would be retained in the micelles during the circulation, but would be released at the low endosomal pH of the tumor cells, upon arrival to the tumor tissue through the EPR effect and/or active targeting and subsequent tumor cell internalization by endocytosis.
Targeted core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin M. Talelli⁎, M. Iman, C.J.F. Rijcken, C.F. van Nostrum, W.E. Hennink Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3508 TB Utrecht, The Netherlands ⁎Corresponding author. E-mail: [email protected]. Abstract summary Core-crosslinked thermosensitive and biodegradable polymeric micelles were actively targeted to EGFR-overexpressing cancer cells by conjugating an anti-EGFR nanobody on the surface of the micelles. A methacrylated doxorubicin derivative containing an acid sensitive hydrazone spacer was encapsulated and subsequently covalently attached during core-crosslinking of the micelles. Encapsulation efficiency was 60% and doxorubicin (DOX) was completely released after 24 h at pH 5, while hardly any DOX was released at pH 7.4. DOXloaded micelles showed toxicity similar to free doxorubicin towards ovarian carcinoma cells. Introduction Conventional anticancer treatments include the use of cytotoxic compounds that kill tumor cells. However, these compounds also inhibit the growth and viability of various healthy cells like bone marrow and gastrointestinal tract cells, causing severe systemic side effects. For such cytotoxic agents, efficient drug delivery systems (DDS) are needed, that specifically deliver these compounds in tumor cells. For intravenous administration of hydrophobic drugs, polymeric micelles are an interesting type of DDS. Thermosensitive and biodegradable polymeric micelles composed of mPEG-pHPMAmlactate have been used in the encapsulation of several anticancer drugs, as well as an MRI contrast agent [1–4]. However, even though
Fig. 2. Cell association (by FACS analysis) of rhodamine micelles (a) and rhodamine nanobody micelles (b), after incubation with A431 cells and concentration dependence of the mean fluorescence intensity.
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Conclusion The results obtained in this preliminary work highlighted that the new complex PPS:chitosan can be easily prepared. Further studies will be carried out in vaginal environment in order to evaluate the influence of natural enzyme in the complex dissolution and in drug release. Acknowledgements The authors wish to thank Bene pharmaChem for the free PPS raw material. References [1] R. Peeker, M. Fall, Int Urogynecol J 11 (2000) 23–32. [2] V.R. Anderson, C.M. Perry, Drugs 66 (2006) 821–835. [3] O. Felt, P. Buri, R. Gurny, Drug Delivery and Industrial Pharmacy 24 (1998) 979–993.
doi:10.1016/j.jconrel.2010.07.090
Novel paclitaxel nanoparticles: Development, in vitro anti-tumor activity in BT-549 cells and in vivo evaluation
Fig. 3. PPS release test.
Gopal V. Shavi⁎, A. Ranjith Kumar, A. Karthik, M. Naseer, G. Aravind, B.D. Praful, M. Sreenivasa Reddy, N. Udupa Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka-576104, India ⁎Corresponding author. E-mail: [email protected]. Abstract summary The aim is to develop paclitaxel PLGA nanoparticles (NPs), for intravenous administration, with improved therapeutic efficacy using single-emulsion technique. Encapsulation efficiency was higher at polymer-to-drug ratio of 20:1 with particle size <200 nm. In vitro drug release exhibited biphasic pattern with initial burst release followed by slow and continuous release for 15 days. In vitro antitumor activity against BT-549 cells showed complete inhibition after 168 h of incubation. AUC and t1/2 were found to be higher for NPs.
Fig. 4. Chitosan release test.
The behavior of PPS release is quite different depending from the medium used. In fact, independently from the PPS content, in phosphate buffer only about 15% of active is released after 96 h instead of more than 50% is released at pH 3. Differently, the amount of chitosan dissolved is quite the same for both medium tested (about 30% after 96 h).
Introduction Paclitaxel (Taxol®) is one of the best antineoplastic agents found from nature in the past decades. Like many other anticancer drugs, there are difficulties in its clinical administration due to its poor solubility. To enhance its solubility and allow parenteral administration, paclitaxel is currently available as 6 mg/ml solution in a vehicle composed of 1:1 blend of cremophor EL and ethanol, but this has been found to cause adverse side-effects such as hypersensitivity reactions, nephrotoxicity, neurotoxicity as well as effects on endothelial and vascular muscles causing vasodilation, labored breathing, lethargy and hypotension. Nanoparticles of biodegradable polymers can provide an ideal solution to the adjuvant problem and realize a controlled delivery by passive targeting of the drug with better efficacy and fewer side-effects. Moreover, nanoparticles can escape from the vasculature through the leaky endothelial tissue that surrounds the tumor and then accumulate in certain solid tumors by the so-called Enhanced Permeation and Retention (EPR) effect. A cremophor free formulation of paclitaxel nanoparticles, Abraxane®, has been approved by FDA for recurrent metastatic breast cancer. Polymeric nanoparticles demonstrated that a significant improvement in drug specificity of action, this effect being mainly attributed to changes in tissue distribution and pharmacokinetics. The objective of this study was to develop a polymeric drug delivery system for paclitaxel with PLGA 50:50 intended to be intravenously administered, capable of improving the therapeutic index of the drug and devoid of the adverse effects of cremophor EL. In this study, the paclitaxel was encapsulated within PLGA 50:50
e120
Abstracts / Journal of Controlled Release 148 (2010) e112–e124
using poly vinyl alcohol (PVA) as emulsifier, to stabilize the dispersed oil droplets which create a sustained-release system over a period of hours to days with improved anti-tumoral efficacy. Experimental methods Nanoparticles were prepared by simple interfacial deposition technique with different ratio of PLGA 50:50 to drug, (5:1, 10:1, 15:1 and 20:1) were dissolved in a certain amount (0.5 ml) of dichloromethane. The resulting solution, (organic phase) was added slowly to the aqueous phase containing PVA solution (1.0% w/v), homogenized using high speed homogenizer with a speed of 15,000 rpm followed by sonication in ice water bath for 3 min at 10 kV using probe sonicator to form nanoparticles. The resulting emulsion was kept under stirring up to 12 h for complete evaporation of organic solvent, centrifuged at 15,000 rpm for 30 min for separation of nanoparticles, obtained as the pellet further dispersed in mannitol solution (5 mg/ml) as cryoprotectant and lyophilized for 48 h at −40 °C with a vacuum pressure of 50 mm Torr to obtain dry fluffy mass. Compatibility studies of drug and carrier system were carried out using Differential Scanning Calorimetry (DSC) analysis. The influence of different experimental parameters such as polymer to drug ratio, organic to aqueous phase ratio, homogenization speed and sonication time on the encapsulation efficiency of paclitaxel in the nanoparticles was evaluated. Nanoparticles were evaluated for percentage encapsulation efficiency (amount of drug encapsulated in the nanoparticles was analyzed by HPLC method), particle size, polydispersity index, zeta potential, surface morphology and in vitro drug release studies using vial method. The effect of pure drug and paclitaxel nanoparticles (NPs) on the viability of BT-549 cells (breast cancer cells) was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The cells were incubated with the pure drug and paclitaxel nanoparticles suspension at concentrations of 0.01, 0.1, 1 and 10 μg/ml, respectively. At specific intervals of 24, 48, 72 and 168 h % cell viability was measured by a microtiter plate reader. Accelerated stability studies at 25 ± 2 °C and 60% ± 5% RH were carried out for the optimized formulation (20:1) for a period of 180 days. Pharmacokinetics of the pure drug and paclitaxel nanoparticles were carried out in Wistar rats (5 mg/kg) and the serum samples were analyzed using HPLC. Pharmacokinetic parameters i.e. AUC, elimination rate constant (ke), t1/2 and clearance (Cl) were determined and data obtained was statistically analyzed by using independent sample t-test with SPSS software 11.5 version, to stumble out significant differences between two groups. Statistical significance was established at p < 0.05.
Paclitaxel-loaded biodegradable drug delivery systems manufactured from single emulsion technique with PLGA 50:50 were reproducible with highest encapsulation efficiency (94.5 ± 4.2) for the polymer to drug ratio of 20:1. The particle size, polydispersity index and zeta potential were found to be in the range of 150–200 nm, 0.257– 0.425 and −33.5 to −68.9 respectively. The preparation allowed the formation of spherical nanometric, homogeneous and negatively charged zeta potential, proved no aggregation and sedimentation with good polydispersity index. Surface morphology studies revealed smooth and spherical nature of the particles as shown in Fig. 2.
Fig. 2. Surface morphology of the polymeric nanoparticles.
The release behavior of paclitaxel from the developed nanoparticles exhibited a biphasic patter characterized by an initial fast release during the first 24 h, followed by a slower and continuous at extremely slow rates for a period of 15 days. The results of cell viability are shown in Fig. 3a and b, after 24 h of incubation time, no cytotoxic effect could be observed for any of the concentrations tested. However for each of the concentrations there was an enhancement in cytoxicity with increasing time of incubation. Cell growth was completely inhibited for paclitaxel nanoparticles after 168 h of incubation when compared to pure drug. Results of the stability studies revealed that the prepared nanoparticles are stable at 25 ± 2 °C and 60% ± 5% RH for a period of 180 days. The results of pharmacokinetic parameters are shown in Table 1. The values area under the concentration-versus-time curve (AUC) and t1/2 of paclitaxel NPs were found to be much higher (3–4 times) for paclitaxel vectored by PLGA (50:50) nanoparticles than for pure drug. Nanoparticles were relatively long circulating (t1/2 33.19 h) with low clearance rate when compared to pure drug.
Results and discussion The selection of an optimal formulation in the study was based on that which provided a combination of better morphology (in terms of sphericity and discreteness), extreme unaffected particle sizes and drug entrapment. DSC analysis indicated the absence of interaction between the drug and selected polymer and excipient, the same was shown in Fig.1.
Fig. 1. DSC analysis of a) paclitaxel and b) polymeric nanoparticles.
Fig. 3. a. Cell viability of BT-549 cells incubated with pure drug. b. Cell viability of BT549 cells incubated with paclitaxel NPs.
Abstracts / Journal of Controlled Release 148 (2010) e112–e124 Table 1 Pharmacokinetic parameters of pure drug and paclitaxel NPs after i.v. administration into rats. Values Parameter
Unit
Paclitaxel NPs
Pure drug (Taxol®)
AUC Elimination rate constant(Ke) t1/2 Clearance(Cl)
h μg/ml 1/h h L/h kg
19.032 0.0208 33.191 0.748
6.108 0.163 5.003 1.273
e121
these micelles (when core-crosslinked) display prolonged circulation and enhanced passive tumor accumulation due to the EPR effect, the loaded drug is rapidly diffused out after injection in the blood stream. So in this study doxorubicin, a cytotoxic drug, was selected for covalent attachment to the core of these micelles, using a methacrylated derivative of doxorubicin (Fig. 1b).
Conclusion The present study showed controlled delivery of paclitaxel from nanoparticles with enhanced anti-tumoral efficacy. Hence Paclitaxel NPs may be considered as an effective anticancer drug delivery system for long term treatment of breast cancer. Acknowledgements The authors are grateful to Getwell Pharmaceuticals PVT Ltd., Gurgaon, India for providing gift sample of paclitaxel. This study was also supported by Department of Science and Technology (DST), New Delhi, India. References [1] F. Danhier, N. Lecouturier, B. Vroman, C. Jérôme, J. Marchand-Brynaert, O. Feron, V. Préat, Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation, J. Control. Release 133 (2009) 11–17. [2] C. Fonseca, S. Simoes, R. Gaspar, Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity, J. Control. Release 133 (83) (2002) 273–286.
doi:10.1016/j.jconrel.2010.07.091
Fig. 1. (a) Chemical structure of micelle forming methacrylated mPEG-b-pHPMAm-Lacn, (b) methacrylated derivative of doxorubicin with a hydrolyzable hydrazone linker.
This prodrug could be co-crosslinked in the hydrophobic core of the micelles composed of methacrylated block copolymers (Fig. 1a) by means of KPS and TEMED polymerization. Importantly, this doxorubicin derivative contains a biodegradable hydrazone linker, which is stable at physiological pH, but hydrolyzes in acidic environment [5]. Thus it was hypothesized that doxorubicin would be retained in the micelles during the circulation, but would be released at the low endosomal pH of the tumor cells, upon arrival to the tumor tissue through the EPR effect and/or active targeting and subsequent tumor cell internalization by endocytosis.
Targeted core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin M. Talelli⁎, M. Iman, C.J.F. Rijcken, C.F. van Nostrum, W.E. Hennink Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3508 TB Utrecht, The Netherlands ⁎Corresponding author. E-mail: [email protected]. Abstract summary Core-crosslinked thermosensitive and biodegradable polymeric micelles were actively targeted to EGFR-overexpressing cancer cells by conjugating an anti-EGFR nanobody on the surface of the micelles. A methacrylated doxorubicin derivative containing an acid sensitive hydrazone spacer was encapsulated and subsequently covalently attached during core-crosslinking of the micelles. Encapsulation efficiency was 60% and doxorubicin (DOX) was completely released after 24 h at pH 5, while hardly any DOX was released at pH 7.4. DOXloaded micelles showed toxicity similar to free doxorubicin towards ovarian carcinoma cells. Introduction Conventional anticancer treatments include the use of cytotoxic compounds that kill tumor cells. However, these compounds also inhibit the growth and viability of various healthy cells like bone marrow and gastrointestinal tract cells, causing severe systemic side effects. For such cytotoxic agents, efficient drug delivery systems (DDS) are needed, that specifically deliver these compounds in tumor cells. For intravenous administration of hydrophobic drugs, polymeric micelles are an interesting type of DDS. Thermosensitive and biodegradable polymeric micelles composed of mPEG-pHPMAmlactate have been used in the encapsulation of several anticancer drugs, as well as an MRI contrast agent [1–4]. However, even though
Fig. 2. Cell association (by FACS analysis) of rhodamine micelles (a) and rhodamine nanobody micelles (b), after incubation with A431 cells and concentration dependence of the mean fluorescence intensity.