Publicação
Liposomes as nanosystems for the transport and delivery of bioactive agents
| Resumo: | The concept of pharmaceutical nanobiotechnology was originated in the 1970s when liposomes started to be used as nano-drug delivery systems (NanoDDS) to incorporate lipophilic and hydrophilic drugs. Since then liposomes have been the most widely investigated nano-carrier system aiming to achieve controlled drug delivery. The inability of several conventional therapies to deliver the therapeutic dose of the active agents to the diseased tissues at the desired time and concomitantly avoid causing severe toxic effects to healthy tissues or organs has brought considerable attention to the development and clinical use of NanoDDS. In the work developed in this thesis we intended to use liposomes as NanoDDS for cytosolic delivery of bioactive agents aiming to target either diseases of the Mononuclear Phagocytic System (MPS) or cancer. Simultaneously we explored the flexibility and the multifunctional nature of liposomes in different aspects. Special attention was given on the potential of liposomes to carry new bioactive agents with distinctive physicochemical features: small molecules (anti-parasitic drugs) or macromolecules (oligonucleotides) and their ability to target different types of cells, such as macrophages (phagocytic cells) and tumour cells (non-phagocytic). In order to achieve our aims we chose two disease models: Leishmaniasis and Small Cell Lung Cancer (SCLC). The development of liposomal formulations of dinitroanilines for the treatment of leishmaniasis was addressed in Chapter II. Dinitroanilines have proved in vitro anti-leishmanial activity but they are not used in clinical practice as chemotherapeutics for the treatment of leishmaniasis. Nevertheless, they hold great potential in the treatment of this disease due to a selective mechanism of action against parasite tubulins and to the absence of toxicity to mammals. To reach this aim we chose two complementary strategies. The first (Part A) consisted in the association of one dinitroaniline, trifluralin (TFL) with conventional liposomes. The second (Part B) consisted in the incorporation of chemical derivatives of TFL (TFL-D) prepared by organic chemistry hemi-synthesis methods, in order to further improve the chemical stability and biological activity. Conventional liposomes were used as solvents for these hydrophobic and difficult to handle dinitroanilines (either the TFL or the TFL-D) and also because they are naturally cleared from the circulation by the MPS favouring their choice to target intracellular infections of this system, such as leishmaniasis. xiv In Part A, after achieving an efficient incorporation and stabilization of TFL in liposomal formulations (stability on storage up to 2 years in lyophilized form) their therapeutic activity in appropriated animal models of visceral and cutaneous leishmaniasis was evaluated. All TFL liposomal formulations were active against different strains of Leishmania, showing significant reduction in the levels of visceral and cutaneous infections in mice. A superior activity (at least 2-fold) was observed for liposomal TFL as compared to the free drug. A selected TFL liposomal formulation also improved the clinical condition of experimentally infected dogs and reduced the parasite load. In Part B, two new dinitroaniline derivatives were used. This approach was pursued mainly to circumvent several disadvantages of TFL such as unfavourable physicochemical properties and difficulties on handling. Selected conventional liposomes were optimised for the incorporation of these TFL-Ds. The anti-leishmanial activity of TFL-D liposomal formulations was evaluated both in vitro and in vivo. The in vitro biological evaluation of the TFL-D liposomal formulations has demonstrated their activity against Leishmania parasites in culture without revealing signs of toxicity. In addition, extensive parasite load inhibition (> 90%) was observed after treatment with one of the TFL-D liposomal formulations in a murine model of zoonotic visceral leishmaniasis. The use of liposomes as NanoDDS in cancer therapy was addressed in Chapter III. The association of conventional anti-cancer drugs with liposomes has been particularly investigated not only because it increases their concentration in the tumour tissue and reduces their negative side effects, but also because of the extensive application of gene therapy protocols in the treatment of cancer. In fact, antisense oligonucleotides (asODN) or other nucleic acid molecules are considered a new class of anti-cancer drugs since they are able to selectively inhibit the expression of a gene. They act by binding to a complementary region of the mRNA causing its degradation with the consequent down-regulation of the corresponding protein. However, nucleic acids molecules need adequate NanoDDS to be efficiently delivered into the cytosol of the tumour cells due to their poor stability in physiological fluids, high susceptibility to nuclease degradation and limited ability to penetrate through cellular membranes. Based on this rationale, Chapter III is focused on the development of a targeted-liposome delivery system containing an asODN for the treatment of SCLC. For this purpose, long circulating (PEG-grafted) cationic liposomes were used for the encapsulation of the asODN. The attachment of a targeting ligand for selective xv cellular delivery, on the outer surface of this long circulating formulation, makes it a specific delivery system for SCLC cells. Two different cationic liposomal formulations, Coated Cationic Liposomes (CCL) and Stabilized Antisense Lipid Particles (SALP), were selected for the encapsulation of an asODN that inhibits the expression of c-myc oncogene, associated with SCLC cell proliferation. The hexapeptide antagonist G was chosen as the targeting ligand to promote internalization of these formulations. The effect of the peptide coupling method, conventional and post-insertion, on the loading capacity and size of both formulations was assessed. The post-insertion coupling method applied both to CCL and SALP liposomes containing as(c-myc), developed in Chapter III resulted in antagonist G-targeted formulations with the necessary characteristics for evaluation of in vitro delivery of asODN to SCLC. The strategy of using antagonist G as the targeting ligand proved to be successful as it increased the uptake of both formulations as compared to the non-targeted counterparts, in particular in a variant SCLC cell line characterised by being resistant to conventional chemotherapy. The presence of the antagonist G at the surface of SALP did not affect the long circulation characteristics of the SALP liposomes as shown in pharmacokinetic studies. In addition, the preferential accumulation of this formulation in the lungs, substantiate the rationale behind the design of these targeted liposomes for in vivo intracellular delivery of nucleic acids. Overall, the main objectives of this work were reached. Throughout its experimental development new and important issues were identified and remain open. These issues may be an interesting starting point for future research. |
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| Autores principais: | Carvalheiro, Manuela |
| Assunto: | Teses de doutoramento - 2011 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The concept of pharmaceutical nanobiotechnology was originated in the 1970s when liposomes started to be used as nano-drug delivery systems (NanoDDS) to incorporate lipophilic and hydrophilic drugs. Since then liposomes have been the most widely investigated nano-carrier system aiming to achieve controlled drug delivery. The inability of several conventional therapies to deliver the therapeutic dose of the active agents to the diseased tissues at the desired time and concomitantly avoid causing severe toxic effects to healthy tissues or organs has brought considerable attention to the development and clinical use of NanoDDS. In the work developed in this thesis we intended to use liposomes as NanoDDS for cytosolic delivery of bioactive agents aiming to target either diseases of the Mononuclear Phagocytic System (MPS) or cancer. Simultaneously we explored the flexibility and the multifunctional nature of liposomes in different aspects. Special attention was given on the potential of liposomes to carry new bioactive agents with distinctive physicochemical features: small molecules (anti-parasitic drugs) or macromolecules (oligonucleotides) and their ability to target different types of cells, such as macrophages (phagocytic cells) and tumour cells (non-phagocytic). In order to achieve our aims we chose two disease models: Leishmaniasis and Small Cell Lung Cancer (SCLC). The development of liposomal formulations of dinitroanilines for the treatment of leishmaniasis was addressed in Chapter II. Dinitroanilines have proved in vitro anti-leishmanial activity but they are not used in clinical practice as chemotherapeutics for the treatment of leishmaniasis. Nevertheless, they hold great potential in the treatment of this disease due to a selective mechanism of action against parasite tubulins and to the absence of toxicity to mammals. To reach this aim we chose two complementary strategies. The first (Part A) consisted in the association of one dinitroaniline, trifluralin (TFL) with conventional liposomes. The second (Part B) consisted in the incorporation of chemical derivatives of TFL (TFL-D) prepared by organic chemistry hemi-synthesis methods, in order to further improve the chemical stability and biological activity. Conventional liposomes were used as solvents for these hydrophobic and difficult to handle dinitroanilines (either the TFL or the TFL-D) and also because they are naturally cleared from the circulation by the MPS favouring their choice to target intracellular infections of this system, such as leishmaniasis. xiv In Part A, after achieving an efficient incorporation and stabilization of TFL in liposomal formulations (stability on storage up to 2 years in lyophilized form) their therapeutic activity in appropriated animal models of visceral and cutaneous leishmaniasis was evaluated. All TFL liposomal formulations were active against different strains of Leishmania, showing significant reduction in the levels of visceral and cutaneous infections in mice. A superior activity (at least 2-fold) was observed for liposomal TFL as compared to the free drug. A selected TFL liposomal formulation also improved the clinical condition of experimentally infected dogs and reduced the parasite load. In Part B, two new dinitroaniline derivatives were used. This approach was pursued mainly to circumvent several disadvantages of TFL such as unfavourable physicochemical properties and difficulties on handling. Selected conventional liposomes were optimised for the incorporation of these TFL-Ds. The anti-leishmanial activity of TFL-D liposomal formulations was evaluated both in vitro and in vivo. The in vitro biological evaluation of the TFL-D liposomal formulations has demonstrated their activity against Leishmania parasites in culture without revealing signs of toxicity. In addition, extensive parasite load inhibition (> 90%) was observed after treatment with one of the TFL-D liposomal formulations in a murine model of zoonotic visceral leishmaniasis. The use of liposomes as NanoDDS in cancer therapy was addressed in Chapter III. The association of conventional anti-cancer drugs with liposomes has been particularly investigated not only because it increases their concentration in the tumour tissue and reduces their negative side effects, but also because of the extensive application of gene therapy protocols in the treatment of cancer. In fact, antisense oligonucleotides (asODN) or other nucleic acid molecules are considered a new class of anti-cancer drugs since they are able to selectively inhibit the expression of a gene. They act by binding to a complementary region of the mRNA causing its degradation with the consequent down-regulation of the corresponding protein. However, nucleic acids molecules need adequate NanoDDS to be efficiently delivered into the cytosol of the tumour cells due to their poor stability in physiological fluids, high susceptibility to nuclease degradation and limited ability to penetrate through cellular membranes. Based on this rationale, Chapter III is focused on the development of a targeted-liposome delivery system containing an asODN for the treatment of SCLC. For this purpose, long circulating (PEG-grafted) cationic liposomes were used for the encapsulation of the asODN. The attachment of a targeting ligand for selective xv cellular delivery, on the outer surface of this long circulating formulation, makes it a specific delivery system for SCLC cells. Two different cationic liposomal formulations, Coated Cationic Liposomes (CCL) and Stabilized Antisense Lipid Particles (SALP), were selected for the encapsulation of an asODN that inhibits the expression of c-myc oncogene, associated with SCLC cell proliferation. The hexapeptide antagonist G was chosen as the targeting ligand to promote internalization of these formulations. The effect of the peptide coupling method, conventional and post-insertion, on the loading capacity and size of both formulations was assessed. The post-insertion coupling method applied both to CCL and SALP liposomes containing as(c-myc), developed in Chapter III resulted in antagonist G-targeted formulations with the necessary characteristics for evaluation of in vitro delivery of asODN to SCLC. The strategy of using antagonist G as the targeting ligand proved to be successful as it increased the uptake of both formulations as compared to the non-targeted counterparts, in particular in a variant SCLC cell line characterised by being resistant to conventional chemotherapy. The presence of the antagonist G at the surface of SALP did not affect the long circulation characteristics of the SALP liposomes as shown in pharmacokinetic studies. In addition, the preferential accumulation of this formulation in the lungs, substantiate the rationale behind the design of these targeted liposomes for in vivo intracellular delivery of nucleic acids. Overall, the main objectives of this work were reached. Throughout its experimental development new and important issues were identified and remain open. These issues may be an interesting starting point for future research. |
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