Biodegradable polymeric nanoparticles (nps) have shown to be promising forms for the delivery of a wide array of drug formulations, because of their ability to: (i) increase the drug half-life in the blood stream, (ii) enhance the solubility of poorly-water soluble drugs, which represents the main obstacle to their efficient administration (iii) improve drug bioavailability and (iv) reduce systemic toxicity. These properties are of particular interest when dealing with deseases like cancer, since they offer the possibility to overcome toxicity and administration problems associated with traditional chemotherapy. Moreover, polymeric nanoparticles are able to passively target tumour cells through the Enhanced Permeation and Retention (EPR) Effect. Tumours are composed of fastly- growing cells which need an extensive supply of oxygen and other nutrients. Their vessels are therefore highly fenestrated (200-300nm) and allow small-size particles to accumulate inside cancer cells. Moreover tumours lack of an efficient lymphatic system and are not able to eliminate these particles once they entered the cells. In this thesis work, biodegradable polymeric nanoparticles, prepared with different materials and techniques were proposed for the active and passive targeting of cancer cells and for the functionalization of biomimetic constructs for tissue engineering. The first section of the present work was mainly focused on the preparation of nanoparticles starting from preformed polyesterurethanes (PU) through two widely used and highly reproducible techniques. Chapter two describes the preparation of nanoparticles by the Solvent Displacement method with two widely used commercial polyesters, poly(ε-caprolactone) and poly(D,L lactide) and the newly synthesized polyesterurethane C-BC2000, based on poly(ε-caprolactone) blocks. The solvent displacement method was selected, since it leads to the formation of monodisperse, small particles by simple precipitation of the polymer in the non solvent (water) without the use of toxic solvents or the application of shear stresses. Paclitaxel, one of the most potent antineoplastic drugs, was encapsulated inside the carriers, which were analyzed in terms of: Morphology, by means of scanning electron microscopy (SEM) and atomic force microscopy (AFM) Size, size distribution, zeta potential xix Encapsulation efficiency and release properties Hydrolytic degradation pathways Uptake by A431 human epidermal carcinoma cells Results obtained for PU nanoparticles, regarding morphology, size, size distribution and surface charge are comparable to those observed with commercial polyesters. All particles possess a small size (<250nm), a low PDI, indicating a narrow size distribution and a regular spherical shape, with little coalescence. In addition, polyurethane nanoparticles exhibited superior properties, such as longer and more controlled release kinetics of the encapsulated drug and a higher cellular internalization. In fact, C-BC2000 nanoparticles had the highest encapsulation efficiency (89±2%) compared to only 24±7% and 18±8% for PLA and PCL, respectively. Moreover the release profile for particles prepared with commercial polyesters, showed that almost all the encapsulated drug was released within the first 48 hours and no further release was observed after this time-point. C-BC2000 nanoparticles were able to sustain the release of the drug for longer time (up to 5 days), when 30% of the active principle was released, indicating that the therapeutic effect of these carriers could last even longer. In chapter 3 two different polyesterurethanes, synthesized with different chain extenders (named C-BC2000 and NS-BC2000) and three commercial polyesters (PCL, PLA and poly(D,L lactide-co-glycolide) PLGA) were used to prepare Paclitaxel-loaded nanoparticles for the active targeting of breast cancer. A modified Single Emulsion Solvent Evaporation method was used, by selecting ethyl acetate as internal phase, Poly(vinyl alcohol) (PVA) as emulsifier and water as external phase. Particles were characterized in terms of morphology, size distribution and zeta potential. Carriers with good spherical shape, small size (<200nm) and negative surface charge were obtained, regardless of the polymer used as matrix-forming material. To augment the targeting ability of nanoparticles towards breast cancer cells, Herceptin, a monoclonal antibody able to selectively recognize a specific receptor (HER2) over- expressed on 25-30% of invasive breast cancer, was attached to the surface of the carriers by hydrophilic/hydrophobic interactions. Surface coating was successfully achieved, as shown by the changes in zeta potential detected for all carriers. xx Moreover the ability of coated particles to selectively recognize their target was tested, by using two different breast cancer cell lines: MCF7, which show a normal expression of the HER2 receptor, and SKBR3 which are known to highly over-express the same receptor. Quantitative uptake studies showed good cellular internalization, by both cell lines and an augmented uptake by SKBR3 cells, when the surface was coated with the monoclonal antibody, confirming that a targeting effect was achieved. Encapsulation efficiency and release profiles from both coated and uncoated particles were also analyzed, and the ability of drug-loaded particles (at three different doses) to effectively kill SKBR3 tumor cells was evaluated. PU showed interesting properties, being superior to commercial polyester in terms of drug release profiles, cellular internalization and cytotoxic activity. In fact, for C-BC2000 and NS-BC2000 nanoparticles, the amount of Herceptin detected on the surface was higher than for the other polymers, except for PCL. However, PCL nanoparticles showed no release of the encapsulated drug and poor cytotoxicity on SKBR3 cells. Moreover, both coated and uncoated PU nanoparticles exhibited the highest cellular uptake and good activity in killing cancer cells, as demonstrated by cytotoxicity tests. The second section of the present work was dedicated to the optimization and characterization of a new, bioactive and biomimetic bone cement containing nanoparticles for the controlled release of an anti-inflammatory agent (Indomethacin). This material is composed of two phases which are naturally present in bones: an organic phase made of chitosan (CH) and collagen as well as of β tricalcium phosphate as inorganic phase, which is expected to increase bioactivity (i.e. the ability to stimulate new bone deposition) of the material. Chitosan and collagen were selected because of their ability to improve injectability and mechanical strength, respectively. Moreover collagen is expected to confer high biomimetic ability to the material. Genipin (GP) was selected as cross-linker for chitosan, due to its natural origin and biocompatibility. The cement composition was optimized to obtain the desired hardening time and dissolution rate and then reinforced with collagen. Moreover, anti-inflammatory properties were given by including Indometacin-loaded biodegradable nanoparticles based on Poly(ε-caprolactone) (PCL) in the cement composition. Nanoparticles were prepared by the Solvent Displacement Method and their size, encapsulation efficiency as well as release profiles were characterized. xxi Injectability, compressive strength and bioactivity of the final cement were also investigated. The material showed good bioactivity, promoting the formation of apatite after 14 days of incubation in simulated body fluids (SBF), especially when high concentrations of inorganic phase were used. The material also possessed a low setting time (around 20min) and good injectability (95%), making it an optimal candidate as injectable bone substitute. Mechanical strength under compression was also analyzed, resulting in poor properties when collagen is not part of the composition. After the addition of collagen, the Young modulus for compression increased from 0,15Mpa to 1Mpa, which meets the requirements for a good vertebral substitute. Moreover the addition of nanoparticles confers anti-inflammatory properties, as well as additional mechanical strength, since the release rate of the drug is compatible with the infection raising kinetics.

SMART POLYMERIC DRUG NANOCARRIERS FOR BIOMEDICAL APPLICATIONS / Mattu, Clara. - (2012). [10.6092/polito/porto/2501970]

SMART POLYMERIC DRUG NANOCARRIERS FOR BIOMEDICAL APPLICATIONS

MATTU, CLARA
2012

Abstract

Biodegradable polymeric nanoparticles (nps) have shown to be promising forms for the delivery of a wide array of drug formulations, because of their ability to: (i) increase the drug half-life in the blood stream, (ii) enhance the solubility of poorly-water soluble drugs, which represents the main obstacle to their efficient administration (iii) improve drug bioavailability and (iv) reduce systemic toxicity. These properties are of particular interest when dealing with deseases like cancer, since they offer the possibility to overcome toxicity and administration problems associated with traditional chemotherapy. Moreover, polymeric nanoparticles are able to passively target tumour cells through the Enhanced Permeation and Retention (EPR) Effect. Tumours are composed of fastly- growing cells which need an extensive supply of oxygen and other nutrients. Their vessels are therefore highly fenestrated (200-300nm) and allow small-size particles to accumulate inside cancer cells. Moreover tumours lack of an efficient lymphatic system and are not able to eliminate these particles once they entered the cells. In this thesis work, biodegradable polymeric nanoparticles, prepared with different materials and techniques were proposed for the active and passive targeting of cancer cells and for the functionalization of biomimetic constructs for tissue engineering. The first section of the present work was mainly focused on the preparation of nanoparticles starting from preformed polyesterurethanes (PU) through two widely used and highly reproducible techniques. Chapter two describes the preparation of nanoparticles by the Solvent Displacement method with two widely used commercial polyesters, poly(ε-caprolactone) and poly(D,L lactide) and the newly synthesized polyesterurethane C-BC2000, based on poly(ε-caprolactone) blocks. The solvent displacement method was selected, since it leads to the formation of monodisperse, small particles by simple precipitation of the polymer in the non solvent (water) without the use of toxic solvents or the application of shear stresses. Paclitaxel, one of the most potent antineoplastic drugs, was encapsulated inside the carriers, which were analyzed in terms of: Morphology, by means of scanning electron microscopy (SEM) and atomic force microscopy (AFM) Size, size distribution, zeta potential xix Encapsulation efficiency and release properties Hydrolytic degradation pathways Uptake by A431 human epidermal carcinoma cells Results obtained for PU nanoparticles, regarding morphology, size, size distribution and surface charge are comparable to those observed with commercial polyesters. All particles possess a small size (<250nm), a low PDI, indicating a narrow size distribution and a regular spherical shape, with little coalescence. In addition, polyurethane nanoparticles exhibited superior properties, such as longer and more controlled release kinetics of the encapsulated drug and a higher cellular internalization. In fact, C-BC2000 nanoparticles had the highest encapsulation efficiency (89±2%) compared to only 24±7% and 18±8% for PLA and PCL, respectively. Moreover the release profile for particles prepared with commercial polyesters, showed that almost all the encapsulated drug was released within the first 48 hours and no further release was observed after this time-point. C-BC2000 nanoparticles were able to sustain the release of the drug for longer time (up to 5 days), when 30% of the active principle was released, indicating that the therapeutic effect of these carriers could last even longer. In chapter 3 two different polyesterurethanes, synthesized with different chain extenders (named C-BC2000 and NS-BC2000) and three commercial polyesters (PCL, PLA and poly(D,L lactide-co-glycolide) PLGA) were used to prepare Paclitaxel-loaded nanoparticles for the active targeting of breast cancer. A modified Single Emulsion Solvent Evaporation method was used, by selecting ethyl acetate as internal phase, Poly(vinyl alcohol) (PVA) as emulsifier and water as external phase. Particles were characterized in terms of morphology, size distribution and zeta potential. Carriers with good spherical shape, small size (<200nm) and negative surface charge were obtained, regardless of the polymer used as matrix-forming material. To augment the targeting ability of nanoparticles towards breast cancer cells, Herceptin, a monoclonal antibody able to selectively recognize a specific receptor (HER2) over- expressed on 25-30% of invasive breast cancer, was attached to the surface of the carriers by hydrophilic/hydrophobic interactions. Surface coating was successfully achieved, as shown by the changes in zeta potential detected for all carriers. xx Moreover the ability of coated particles to selectively recognize their target was tested, by using two different breast cancer cell lines: MCF7, which show a normal expression of the HER2 receptor, and SKBR3 which are known to highly over-express the same receptor. Quantitative uptake studies showed good cellular internalization, by both cell lines and an augmented uptake by SKBR3 cells, when the surface was coated with the monoclonal antibody, confirming that a targeting effect was achieved. Encapsulation efficiency and release profiles from both coated and uncoated particles were also analyzed, and the ability of drug-loaded particles (at three different doses) to effectively kill SKBR3 tumor cells was evaluated. PU showed interesting properties, being superior to commercial polyester in terms of drug release profiles, cellular internalization and cytotoxic activity. In fact, for C-BC2000 and NS-BC2000 nanoparticles, the amount of Herceptin detected on the surface was higher than for the other polymers, except for PCL. However, PCL nanoparticles showed no release of the encapsulated drug and poor cytotoxicity on SKBR3 cells. Moreover, both coated and uncoated PU nanoparticles exhibited the highest cellular uptake and good activity in killing cancer cells, as demonstrated by cytotoxicity tests. The second section of the present work was dedicated to the optimization and characterization of a new, bioactive and biomimetic bone cement containing nanoparticles for the controlled release of an anti-inflammatory agent (Indomethacin). This material is composed of two phases which are naturally present in bones: an organic phase made of chitosan (CH) and collagen as well as of β tricalcium phosphate as inorganic phase, which is expected to increase bioactivity (i.e. the ability to stimulate new bone deposition) of the material. Chitosan and collagen were selected because of their ability to improve injectability and mechanical strength, respectively. Moreover collagen is expected to confer high biomimetic ability to the material. Genipin (GP) was selected as cross-linker for chitosan, due to its natural origin and biocompatibility. The cement composition was optimized to obtain the desired hardening time and dissolution rate and then reinforced with collagen. Moreover, anti-inflammatory properties were given by including Indometacin-loaded biodegradable nanoparticles based on Poly(ε-caprolactone) (PCL) in the cement composition. Nanoparticles were prepared by the Solvent Displacement Method and their size, encapsulation efficiency as well as release profiles were characterized. xxi Injectability, compressive strength and bioactivity of the final cement were also investigated. The material showed good bioactivity, promoting the formation of apatite after 14 days of incubation in simulated body fluids (SBF), especially when high concentrations of inorganic phase were used. The material also possessed a low setting time (around 20min) and good injectability (95%), making it an optimal candidate as injectable bone substitute. Mechanical strength under compression was also analyzed, resulting in poor properties when collagen is not part of the composition. After the addition of collagen, the Young modulus for compression increased from 0,15Mpa to 1Mpa, which meets the requirements for a good vertebral substitute. Moreover the addition of nanoparticles confers anti-inflammatory properties, as well as additional mechanical strength, since the release rate of the drug is compatible with the infection raising kinetics.
2012
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11583/2501970
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