Francisco B.T. Pessine Depto. F sico Qu mica

1. Francisco B.T. PessineDepto. Físico Química/Instituto de Química/UNICAMPCP 615413084-971 Campinas/SPe-mail: fpessine@iqm.unicamp.br Encapsulação e caracterização de fármacos em sistemas micro e nano particulados Lipossomas Ciclodextrinas Microesferas Nanoesferas

2. SISTEMAS CARREADORES DE FÁRMACOS PRINCIPAIS APLICAÇÕES DE TECNOLOGIAS MODERNAS PARA ENTREGA DE ATIVOS I. LIPOSSOMAS Adjuvantes em vacinas Entrega sustentável de ativos no local da injeção Entrega passiva a tumores Entrega passiva ao endotélio pulmonar em terapia gênica Entrega a nódulos linfáticos e a outros órgãos II. Niossomas Adjuvantes em vacinas Entrega passiva a tumores III. Nano/Micropartículas Adjuvantes em vacinas Entrega passiva a tumores IV.Emulsões Veículos para administração de ativos lipofílicos V. Ciclodextrinas Solubilização de ativos hidrofóbicos para uso oral e parenteral

3. Lipossomas

4. Lipídios

6. Encapsulação de Nistatina em lipossomas Estrutura molecular

7. Extrusão Tipos de vesículas

8. Tipos de bicamadas

9. Lipossomas Localização de moléculas em bicamadas

10. Lipossomas Formação de um lipossoma

11. Lipossomas Corte longitudinal de um lipossoma

12. Anfotericina B e Nistatina Fórmulas estruturais de AnfB e Nys

13. Molécula de fosfatidilcolina

14. Transferência do conteúdo lipossomal para o interior da célula

15. Inserção de AnfB em bicamada lipossomal

16. Francisco B.T. PessineInstituto de QuímicaDepartamento de Físico Químicafpessine@iqm.unicamp.br ENCAPSULAÇÃO E CARACTERIZAÇÃO DE FÁRMACOS EM SISTEMAS MICRO E NANO PARTICULADOS Lipossomas Ciclodextrinas Micro/Nanoesferas

17. Spectrofluorimetry and Chemometrics for Investigation of Norfloxacin Distribution in Multilamellar Liposomes Applied Spectroscopy xx, xxx (2005) 

18. Introdução Norfloxacin: partially hydrophobic fluoroquinolone antibiotic active against Gram+/- bacteria, inhibiting the gyrase enzyme Topoisomerase II. Good sensitiser of singlet oxygen. Highly effective in the treatment of infectious diseases, mainly in the urinary/respiratory tracts. Has a zwitterionic amphoteric nature, being ionized over the whole pH range, due to -COOH and –NH2 groups. This influence its distribution/affinity for lipid bilayers and it is also related to the phototoxic properties of NFX, under solar UV radiation (induction of dermic tumors in rats, phototoxicity in mamalian cells in vitro, accumulation in lysosomes of HS68 human skin fibroblasts and other citoplasmatic organelles) Entrapment of NFX in liposomes: can be of therapeutic interest as they provide different pathways of interaction with bacterial cells, compared to the normal routes, being useful in the treatment of infections caused by quinolone resistant/poorly sensitive bacteria to this drug. Investigation on the distribution of NFX in liposomes: provides a better understanding of its interaction with biological membranes. .

19. Equilíbrios entre diferentes espécies de NFX

20. Objetivos Investigar a distribuição de NFX em bicamadas lipídicas de lipossomas MLV em pH 7.0 usando: supressão de fluorescência grau de anisotropia quimiometria

21. Experimental MLV containing NFX: prepared with soy-bean phosphatidylcholine (Epikuron 200SH; 25/45mg), cholesterol (15mg) and NFX (10.0?mol) all dissolved in CHCl3:MeOH (2:1; v:v) The solvents were evaporated and the lipid film was hydrated with 10.0mL of 0.010 mol/L HEPES buffer (pH 7.0) at 65oC.

22. Steady state measurements of fluorescence anisotropy of free and liposomal NFX (pH 7.0) were obtained on a Jobin Yvon-Spex Spectrofluorimeter, with L geometry. This technique is based on the excitation and emission of polarized light which excites the fluorophores, according to their orientation relative to the direction of the polarized excitation. The emission can be depolarized by rotational diffusion, with an angular displacement during the lifetime of the excited state. The emitted radiation does not show the same orientation as the excitation one. The degree of anisotropy is:

23. Excitation source: Xe lamp (?=284nm). Fluorescence monitored at 426nm at constant T. Free and encapsulated NFX incubated in quartz cells at 25oC (<Tm?53oC) in the presence of I- and acrylamide as quenchers in 0.010mol/L HEPES buffer (pH 7.0). All solutions contained 0.010mol/L sodium thiophosphate to prevent oxidation of I- toward I-3. Data were analyzed according to Stern-Volmer equation: F0/F: fluorescence intensities in the absence/presence of quencher. KD: Stern-Volmer collision constant. To distinguish between two populations of fluorophores a modified Stern-Volmer equation was used: Fa: fraction of the initial fluorescence supressed by the quencher Q; ?F: difference between the fluorescence intensity in the absence (F0) and presence (F) of quencher; KD: Stern-Volmer constant. 

24. Quimiometria To resolve the overlapped spectra and quenching profiles of the zwitterionic and neutral forms, the chemometric method “Self-Modeling Curve Resolution” was applied. This method uses Principal Components Analysis, based on kernel Singular Value Decomposition (SVD). It allows spectral deconvolution assuming the presence of only two substances. The SMCR method uses the following assumptions: a) the curves must be non-negative b) the curves in the data set must be a linear combination of two linearly independent curves c) at least one wavelength must exist for each substance where just that substance fluoresces. The SMCR method was carried out on a matrix constructed of relative fluorescence spectra, F0/?F. 

25. Resultados/Discussão Anisotropy measurements give qualitative information on the incorporation of NFX molecules into the lipidic bilayers of liposomal vesicles. Fluorescence quenching spectra treated with chemometric methods confirm the existence of two populations of NFX (neutral and zwitterionic) in MLV liposomes at pH 7. Results: the zwitterionic form of NFX was incorporated into lipid/aqueous interface of the liposomes, and the neutral form was located toward the center of the bilayers. I- can quench the fluorescence of both forms of NFX in non-liposomal solution, but it cannot quench the encapsulated neutral form in the bilayer because these ions doesn't have access to the interior of the bilayers. On the other hand, acrylamide can penetrate into the bilayer, reaching the neutral species and quenching its fluorescence.

26. Anisotropy measurements gives a strong indication of encapsulation. The results (Table I), allowed the observation of values directly related to the kind of environment where the fluorophores were distributed. In hydrophobic environments, such as liposomal lipidic bilayers, higher values were noted, followed by ones from micelles. This means that the molecules of NFX penetrate into these two systems, causing a difference in their rotational diffusion, having a more restricted degree of freedom in organized systems like liposomes and miceles. . Free rotations in solution were related to the smallest values of r. However, NFX showed a stronger interaction in EtOH and CH3Cl than in aqueous solutions, decreasing its rotational movements, and increasing the anisotropy values. NFX molecules are inserted deeply into the lipidic bilayer of MLV liposomes. In neutral medium, there is an equilibrium between the neutral and zwitterionic forms of NFX, where the latter one predominates. The neutral form is incorporated into the bilayer, while the zwitterionic form is in contact with the water/lipid interface 

27. Tabela 1: Graus de anisotropia Solutions of NFX (1.0 x 10-5 mol/L) r Deionized water 0.019 HEPES buffer (pH 7) 0.024 Citrate buffer (pH 3) 0.025 Ethanol 0.077 Chloroform 0.052 SDS (pH 7) 0.086 SDS (pH 3) 0.106 MLV (45mg PC; NFX 1.0x10-4mol/L) 0.378 MLV (25mg PC; NFX 1.0x10-4mol/L) 0.269

28. Fluorescence quenching using I- and acrylamide as quenchers, in free and in liposomal NFX solutions (pH=7): the concentrations of quenchers varied from 0 to 0.25mol/L (these concentrations does not changes the bilayer structure). Using Chemometrics: it was possible to resolve the emission spectra and the fluorescence quenching profiles of the neutral/zwitterionic forms of NFX separately (Figs. 2 and 3). The quenching constants were obtained from the experimental spectra and not from the calculated ones. Stern-Volmer equation: applied for each fluorescence quenched spectra. It describes the occurrence of dynamic and static quenching. The Stern-Volmer plots deviated from linearity with upward curvatures, suggesting the presence of static and dynamic quenching. Dynamic quenching: quenchers interacts with the excited state, decreasing the fluorescence intensity and lifetime. Static quenching: the quencher remove the molecules from the excited state, decreasing only the emission intensity. 

29. Fig. 2: Espectros resolvidos e supressão da fluorescência

30. Fig. 3: Espectros resolvidos e supressão da fluorescência

31. Linear Stern-Volmer plot: indicative of a single class of fluorophores, all equally accessible to the quencher. The existence of two populations of fluorophores (neutral and zwitterionic forms) in MLV liposomes at pH 7, which is in accordance with the partial hydrophobicity of NFX, allowed the use of the modified Stern-Volmer equation to obtain the rate of collisional encounters between the fluorophore and the quencher, called Stern-Volmer constant values (Table 2).

32. Tabela 2: Constantes de Stern-Volmer Sample Quencher NFX specie KD (L/mol) NFX I Zwitterion 71.8 Neutral 21.8 NFX-MLV I Zwitterion 14.4 Neutral NFX Acrylamide Zwitterion 2.01 Neutral 3.32 NFXMLV Acrylamide Zwitterion 3.07 Neutral 4.82

33. Acrylamide quencher: it was observed that, for the free drug in solution, the neutral form showed better interaction with this quencher than the zwitterionic (Kzwit=2.01 and Kneut=3.32L/mol). Also, a blue shift in the spectrum was observed for the neutral specie. The ground state of this specie has a smaller dipole moment than the excited one. Such a state is better stabilized when interacting with acrylamide, which is a nonpolar molecule. MLV liposomes: both zwitterionic and neutral forms were quenched by acrylamide, confirming the existence of two populations of NFX (neutral and zwitterionic) in lipid bilayers.The local insertion of acrylamide molecules in the lipid bilayer changes the environment around the NFX, increasing its interactions with the quencher molecules. There is an widening of the spectral band for the neutral specie, which is the one that has a better affinity for acrylamide. The descending behavior of quenching profiles proves the existence of such specie, free in solution or inserted in the lipidic bilayers. 

34. Conclusions According to the modified Stern-Volmer constant values (Table 2), it is seen that I- quenches both forms of NFX when it is free in solution. I-: can quench the fluorescence of the encapsulated drug only when the it is in hydrophilic region. Acrylamide: hydrophobic molecule, located inside the bilayer and quench the fluorescence of encapsulated NFX molecules. MLV liposomes: only the zwitterionic form was accessible to the quencher, although at a lower rate than that observed for the free form. The neutral species was not quenched by I- because it was located deeper, near the center of the bilayer. This lack of quenching is due to the inability of the charged and hydrated I- to enter the non polar interior of the liposome. The drug NFX is incorporated in MLV bilayers.

35. Encapsulação e caracterização do antineoplásico Irinotecan (CPT-11) em lipossomasPatente INPI 204.377-7 (09Out02)D.N. Biloti, M.H.A. Santana, F.B.T. Pessine, Lipid membrane with low proton permeability, Biochim. Biophys. Acta 1611, 1 (2003) Interação com albumina do soro humano (HSA) Encapsulação/caracterização em lipossomas estericamente estabilizados

36. Estruturas químicas de derivados da Camptotecina (CPT) Formas lactona e carboxilato

37. Perfis temporais da fluorescência da lactona e carboxilato A: CPT-11 livre B: CPT-11 lipossomal

38. Espectros resolvidos e perfil temporal da fluorescência A: espectros das formas lactona (___) e carboxilato (- - -) B: perfil temporal das formas lactona (___) e carboxilato (- - -)

39. Relação estrutura-atividade de Camptotecinas Atividade antineoplásica 01: grupos aza ? 02: grupos metileno dióxi ? 03: grupos hidroxi-metoxi ?; substituintes volumosos ? 04: substituições ? 05: grupos alquil ? 06: substituições ? 07: anel piridona essencial 08: anel lactona essencial 09: anel de 7 membros estabiliza a lactona 10: anel lactona intacto essencial 11: isômero R é inativo 12: substituições ?

40. Resolução das bandas espectrais via SVD A: bandas espectrais calculadas com o método SVD

41. Domínios e sub-domínios estruturais de HSA HSA: 1 único resíduo de triptofano

42. Espectros de fluorescência de CPT-11 A: Espectro na ausência de HSA B: Espectro na presença de HSA (30mg/mL)

43. Espectros e perfis temporais da fluorescência de Tyr/Trp/subdomínio IIA/HSA em soluções contendo CPT-11 A: Espectros Tyr (- - - - ) e Trp (----) B: Perfis temporais

44. Espectros de fluorescência de CPT-11 em tampão HEPES A: CPT-11 livre B: CPT-11 lipossomal

45. Espectros de fluorescência de CPT-11 em tampão HEPES A: CPT-11 livre: espécies lactona (-----) e carboxilato (- - - - ) B: CPT-11 lipossomal: espécies latona (-----) e carboxilato (- - - - )

46. Permeabilidade de bicamadas a íons H+ Sonda fluorescente: 9-aminoacridina

47. Permeabilidade de bicamadas a íons H+ Sonda fluorescente: 9-aminoacridina

65. Equipe Doutorado Débora N. Biloti (FAPESP): Irinotecan@LPS Milene H. Martins (CNPq): Isotretinoina@LPS Edeilza G. Brescansin (UEM/PICDT): Nistatina@LPS Débora Simoni (PED): Anfotericina B@LPS Ana P.V. Lala (Capes): Nimodipina@CD Fernanda M. Tomé (UNIP): 5-Fluoruracil@CD Rita C.S. Pompei (Biotech): Vancomicina@LPS Mestrado Angélica Cassemiro (Dosage): Clobetasol@CD Adriana Calderini (CNPq): Minoxidil@CD Iniciação Científica Mônica S.A. Oliveira (FAPESP): Hidroquinona@CD Pós Doc Carlos A. Oliveira (UFU): Zn-ftalocianina@LPS Agradecimentos: FAPESP, CNPq, FAEP, Capes, Cristália, EMS, ICN, Labogen, Medley, Stiefel, Degussa, Genzyme