Interactions of progesterone with zwitterionic dipalmitoyl phosphotidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and progesterone concentration by using three non-invasive techniques of Fourier transform infrared (FTIR) spectroscopy, turbidity at 440 nm and differential scanning calorimetry (DSC). The results show that 1mol% of progesterone does not induce a significant change in the shape of thermotropic profile of DPPC. However as progesterone concentration increases, the main transition temperature decreases and phase transition curve broadens. Higher concentrations (12, 18 and 24mol%) also decreased the transition temperature but not as significantly as lower concentrations. The characteristic pretransition of DPPC was completely disappeared upon the addition of progesterone. Progesterone disorders the phospholipid membranes in a concentration dependent manner. Furthermore, low concentrations of progesterone (3, 6 and 9mol%) increase the fluidity of the system but high concentrations (12, 18 and 24mol%) stabilize the membranes by decreasing the mobility of the acyl chains. The opposite effect of progesterone on membrane dynamics of low and high concentrations was also supported by turbidity studies at 440 nm. DSC peaks broaden and shift to lower temperature degrees with increasing concentrations up to 9mol% of progesterone. At 6 and 12mol% of progesterone, the curve contains more than one peak. This indicates the existence of phase separation. The pretransition of liposomes was eliminated for all samples containing progesterone. Analysis of C=O stretching bond in FTIR spectroscopy showed that progesterone does not make any hydrogen bonds with the interfacial region of DPPC liposomes, instead it induces free carbonyl groups in the system. Ester groups were found to be disordered by the addition of progesterone and the effect is profound with 6 and 9mol% concentrations. The head group of liposomes were found to make hydrogen bonding in the vicinity of 3mol% of progesterone in both phases and of 6mol% of progesterone in liquid crystalline phase by infrared spectroscopy of PO- 2 stretching mode. This hydrogen bonding is made either with the hydroxyl group of progesterone or with the water molecules around the head group. With other concentrations of progesterone, there is no evidence of hydrogen bond formation.

Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eliminated. However, despite these successes there are still many diseases of microbial origin that cause tremendous suffering because there are no vaccines or the vaccines available are inadequate. The development of DNA based vaccines and immunostimulatory CpG oligonucleotides (ODNs) as adjuvants offer new possibilities for developing new vaccines. The objectives of this research were to improve the delivery of polynucleotides and oligonucleotides to enhance their potency and to evaluate the feasibility of non-invasive methods for the delivery of vaccines through the skin in order to improve the safety and the ease of administration of human and veterinary vaccines. The results demonstrated that topical administration of plasmids in a lipid-based delivery system (biphasic lipid vesicles [Biphasix]) resulted in gene expression in the draining lymph nodes, as well as induction of antigen specific immune responses in mice. The use of electroporation significantly enhanced both gene expression and immune responses to DNA vaccines in pigs. Prior treatment with electroporation enhanced immune responses to both protein and DNA vaccines indicating that both gene expression and tissue damage are important mechanisms that electroporation uses to enhance immune responses. In addition, the formulation of CpG ODNs in biphasic lipid vesicles (BiphasixTM) called Vaccine-Targeting Adjuvant (VTA) enhanced immune response to protein antigens following systemic and mucosal administration.

El treball descriu, principalment, la preparació de suspensions de liposomes (L) amb diferents fàrmacs encapsulats (ditranol: DT; hidrocortisona: HC; i butirat-propionat d'hidrocortisona:HBP) així com la d'emulsions o/w amb oli de borratja (OB/aigua) estabilitzades amb fosfolípids (F). Aquestes formulacions tenen interès dermatològic, i els mètodes de preparació emprats són directament escalables a la indústria. Prèviament a l'encapsulació dels fàrmacs en liposomes i a la preparació de les emulsions va ser necessari fer els següents estudis: 1.- L'efecte de la concentració de F, del diàmetre dels liposomes i de la concentració de sacarosa, en la liofilització de les vesícules. Els resultats mostren que únicament els liposomes petits (&#8804;100 nm) presentaren molt baixa estabilitat física durant la liofilització, augmentant notablement el seu diàmetre un cop rehidratats.2.- L'estabilitat dels L en hidrogels obtinguts amb Carbopol 940. Es va comprovar que tots els L estudiats van ser estables en els esmentats hidrogels.3.- La determinació de les condicions d'anàlisi de la grandària de les vesícules mitjançant detecció heterodina de l'espectre de freqüències de la llum dispersada. L'instrument emprat va ser el Microtrac UPA 150 (UPA). Pel que fa a aquesta tècnica, es comprovà que les anàlisis de les grandàries amb l'UPA 150 han de complir les següents condicions:a) un temps de lectura &#8805; 10 minuts; b) un índex de càrrega situat entre 0,1 i 1; c) la necessitat de treballar dins l'interval lineal de la relació concentració de les mostres-índex de càrrega; d) la fracció en volum de la fase contínua ha d'ésser &#8805; 0,97; e) cal realitzar un apantallament de les càrregues elèctriques que puguin tenir les vesícules. En cas contrari, es produeixen alteracions de la seva distribució de velocitats, causant anàlisis incorrectes.4.- La posta a punt de l'obtenció de vesícules amb un homogeneïtzador d'alta pressió escalable a nivell industrial, el Microfluidizer 110S (M110S). Els resultats mostren que el control dels cicles de processament, la pressió de treball, la força iònica del medi aquós de la suspensió, la concentració d'etanol (en cas de formar part de la formulació) i la concentració de F, permeten determinar amb precisió les característiques de la població de L obtinguts amb el M110S. El diàmetre, la dispersió de la població, i el volum encapsulat de les vesícules s'ajusten satisfactòriament a models polinòmics lineals de segon ordre.En referència a l'encapsulció dels fàrmacs en liposomes: · La relació màxima d'encapsulació del DT en L és de 4,74 mg DT/g F (1:60 DT:F mol/mol) independentment de la grandària de les vesícules. L'estabilitat química de les preparacions és molt baixa (<7 dies) en qualsevol de les formes de presentació obtingudes (suspensions, liofilitzats, pro-liposomes). La degradació del fàrmac està afavorida per la presència de fosfolípids insaturats. En l'assaig d'irritació in vitro els L amb DT mostren un grau d'irritació lleugerament inferior al causat per una crema comercial de referència. · La relació màxima d'encapsulació de l'HC en L és de 25,8 mg HC/g F (1:19 HC:F mol/mol) independentment de la grandària de les vesícules. Les suspensions mostren inestabilitat física (aprox. 200 dies). Els estudis d'espectrometria d'infraroig (FT-IR) posen de manifest l'establiment d'enllaços d'hidrogen entre els grups OH de l'HC i els grups fosfat dels F, de manera que el fàrmac es situa a la zona polar de les bicapes (B), actuant com a disruptor. En L unilamel·lars obtinguts per fase reversa s'observa una sobresaturació transitòria (aprox. 12 hores) de les B amb HC. La interacció HC-F és feble, i s'observa una ràpida pèrdua del fàrmac en diluir les mostres.· La relació màxima d'encapsulació de l'HBP en L és de 47,9 mg HBP/g F (1:13 HBP:F mol/mol) independentment de la grandària de les vesícules. Les diferents formulacions (suspensions, hidrogels, liofilitzats) presenten una bona estabilitat química, en particular les liofilitzades (aprox. 10 anys). Els estudis d'FT-IR no detecten enllaços d'hidrogen entre l'HBP i els F. L'aplicació de suspensions de L amb HBP en humans provoca l'emblanquiment de la pell com a conseqüència de l'efecte vasoconstrictor del fàrmac, però no produeix cap disminució estadísticament significativa de la inhibició de la urticària de contacte amb nicotinat de metil (mesurat amb velocimetria de làser Doppler). Aquests resultats s'han associat als efectes d'adsorció/desorció d'aigua a l'estrat corni. Els estudis histomètrics amb ratolins tractats amb L amb HBP posen de manifest la producció d'atròfia cutània en diversos teixits, un efecte secundari del fàrmac relacionat amb la seva potència vasoconstrictora. Els resultats confirmen que els L actuen com a vehicle de l'HBP per aplicació tòpica.· S'obtingueren emulsions o/w amb OB al 2 % p/p estabilitzades amb F emprant el M110S. Les preparacions (emulsions, hidrogels i liofilitzats) presenten una bona estabilitat. El tipus de vesícules presents a les emulsions (L i microgotes) depèn de la relació inicial OB/F. / El trabajo describe, principalmente, la preparación de suspensiones de liposomas (L) con diferentes fármacos encapsulados (ditranol: DT; hidrocortisona: HC; y butirato-propionato de hidrocortisona:HBP) así como la de emulsiones o/w con aceite de borrajas (OB/agua) estabilizadas con fosfolípidos (F). Estas formulaciones tienen interés dermatológico y los métodos de obtención utilizados son directamente transferibles a la producción industrial. Previamente a la encapsulación de los fármacos en liposomas y a la preparación de las emulsiones fue necesario realizar los siguientes estudios:1.- El efecto de la concentración de F, del diámetro de los liposomas y de la concentración de sacarosa en la liofilización de las vesículas. Los resultados muestran que únicamente los liposomas pequeños (&#8804;100 nm) tienen poca estabilidad física durante la liofilización, aumentado notablemente su diámetro una vez rehidratados.2.- La estabilidad de los L en hidrogeles obtenidos con Carbopol 940. Se pudo comprobar que todos los liposomas estudiados fueron estables en dicho gel.3.- La determinación de las condiciones de análisis del tamaño de las vesículas mediante la detección heterodina del espectro de frecuencias de la luz dispersada. El instrumento utilizado fue el Microtrac UPA 150 (UPA). En referencia a esta técnica se comprobó que loas análisis de tamaño con el UPA 150 deben cumplir las siguientes restricciones: a) un tiempo de lectura &#8805; 10 minutos; b) un índice de carga situado entre 0,1 i 1; c) la necesidad de trabajar en el intervalo lineal de la relación concentración de les muestras-índice de carga; d) la fracción en volumen de la fase continua debe ser &#8805; 0,97; e) se deben apantallar las cargas eléctricas que puedan tener las vesículas. En caso contrario, se producen alteraciones de su distribución de velocidades, obteniéndose análisis erróneos.4.- La puesta a punto de la obtención de vesículas con un homogenizador a alta presión escalable a nivel industrial, el Microfluidizer 110S (M110S). Los resultados muestran que el control del número de ciclos de procesamiento, la presión de trabajo, la fuerza iónica del medio acuoso de la suspensión, la concentración de etanol (en caso de formar parte de la formulación) y la concentración de F permiten determinar con precisión las características de la población de L obtenidos con el M110S. El diámetro, la dispersión de la población y el volumen encapsulado de las vesículas se ajustan satisfactoriamente a modelos polinómicos lineales de segundo orden.En referencia a la encapsulación de los fármacos en liposomas:· La relación máxima de encapsulación del DT en L es de 4,74 mg DT/g F (1:60 DT:F mol/mol) independientemente del tamaño de las vesículas. La estabilidad química de las preparaciones es muy baja (<7 días) en todas de las formes de presentación obtenidas (suspensiones, liofilizados, pro-liposomas). La degradación del fármaco esta favorecida por la presencia de fosfolípidos insaturados. El ensayo de irritación in vitro muestra que los L con DT causan un grado de irritación levemente inferior al causado por una crema comercial de referencia.· La relación máxima de encapsulación de la HC en L es de 25,8 mg HC/g F (1:19 HC:F mol/mol) independientemente del tamaño de las vesículas. Las suspensiones muestran inestabilidad física (aprox. 200 días). Los estudios de espectrometría de infrarrojo (FT-IR) ponen de manifiesto la existencia de enlaces de hidrógeno entre los grupos OH de la HC y los grupos fosfato de los F, de manera que el fármaco se sitúa en la zona polar de las bicapas (B), inestabilizándolas. En L unilamelares obtenidos por fase reversa se observa una sobresaturación transitoria (aprox. 12 horas) de las B con HC. La interacción HC-F es débil, y se observa una rápida pérdida del fármaco al diluir las muestras.· La relación máxima de encapsulación del HBP en L es de 47,9 mg HBP/g F (1:13 HBP:F mol/mol) independientemente del tamaño de las vesículas. Las diferentes formulaciones (suspensiones, hidrogeles, liofilizados) presenten buena estabilidad química, en particular las liofilizadas (aprox. 10 años). Los estudios de FT-IR no detectan enlaces de hidrógeno entre el HBP y los F. La aplicación de suspensiones de L con HBP en humanos provoca la decoloración de la piel a consecuencia del efecto vasoconstrictor del fármaco, pero no produce una disminución estadísticamente significativa de la inhibición de la urticaria de contacto con nicotinato de metilo (medida por velocimetría de láser Doppler). Estos resultados se han asociado a los efectos de adsorción/desorción de agua en el estrato córneo. Los estudios histométricos con ratones tratados con L con HBP ponen de manifiesto la producción de atrofia cutánea en diversos tejidos, un efecto secundario del fármaco relacionado con su potencia vasoconstrictora. Los resultados confirman que los L actúan como vehículo del HBP para la aplicación tópica.· Se obtuvieron emulsiones o/w con OB al 2 % p/p estabilizadas con F utilizando el M110S. Las preparaciones (emulsiones, hidrogeles y liofilizados) presentan alta estabilidad. El tipos de vesículas presentes en las emulsiones (L y microgotas) dependen de la relación inicial OB/F. / This work describes the preparation of drug-loaded liposome (dithranol: DT; hydrocortisone: HC; hydrocortisone butyrate-propionate: HBP) liposome (L) suspensions and o/w phopholipid stabilized emulsions (borage oil: BO/water). All preparations are of great interest in dermatology, and they were obtained using scale-up methods. In order to obtain the previously described formulations the following studies were performed: 1.- Study of the effect of the F concentration, the L diameter and the sucrose concentration on the physical L stability during lyophilization. The results show that the small L (&#8804;100 nm) have a very low stability during lyophilization.2.- Study of the effect of Carbopol 940 (hydrogel) on the L stability. The study shows that all the L tested were stable in Carbopol 940 hydrogel preparations.3.- Determination of the factors involved on the measurement of the liposome diameter using the "Microtrac UPA 150 (UPA)", an heterodyne detector spectrometer of sample scattered light. Results indicate that UPA vesicle-sizing analysis have to observe the next conditions: a) a minimum run time of 10 minutes; b) a loading index between 0.1 and 1; c) a lineal relation between sample concentration and the loading index; d) a continuous phase volume fraction &#8805; 0.97;e) the vesicle electrical charge have to be screened. Otherwise, velocity distribution is disturbed and the analysis are not correct.4.- Conditions for the preparation of L using a the high-pressure homogenizer "Microfluidizer 110S (M110S)". It was found that, using experimental design methods, the characteristics of the L obtained with the M110S depends on the control of the number of cycles, pressure, ionic strength, ethanol concentration and P concentration. The diameter, dispersion and entrapped volume of L have a good fit with lineal second order polynomial models.Regarding the drug entrapment in liposomes, results indicate that:· The maximum amount of DT entrapped in L has a ratio of 4.74 mg DT/g P (1:60 DT:P mol/mol) and it is not dependent on L diameter. In all preparations (suspensions, lyophilized and pro-liposome) the chemical stability was very low (< 7 days). Drug degradation is increased by unsaturated P. In vitro irritation test show that DT entrapped in L causes less irritation than that produced by a commercial cream.· The maximum amount of HC entrapped in L has a ratio of 25.8 mg HC/g P (1:19 HC:P mol/mol) and it is not dependent on the L diameter. The suspensions are physically unstable (&#8764;200 days). Infrared spectrometry (FT-IR) experiments detect hydrogen bonds between OH groups of drug and P phosphate group. These results suggest that HC is localized in the polar domain of the lipid bilayer and has a disrupter effect. A temporally bilayer supersaturation (&#8764;12 hours) with HC is observed in unilamellar L obtained by reverse phase evaporation. HC-P interactions are weak, and a fast drug desencapsulation is observed when the samples are diluted.·The maximum amount of HBP entrapped in L has a ratio of 47.9mg HBP/g P (1:13 HBP:P mol/mol) and it is not dependent on L diameter. The suspensions, hydrogels and lyophilized samples show chemical stability, specially the last ones (>10 years). FT-IR experiments do not detect any hydrogen bond between drug and P. Topical treatment with humans show a blanching effect on the skin due to the drug vasoconstrictor properties. However, the inhibition of the methyl-nicotinate-induced skin inflammation is not statistically significant (as has been shown for laser Doppler flowmetry). The last results are associated with adsorption/desorption phenomena in the skin stratum corneum. On the other hand, hystometric results of L-drug treated skin mouse show the atrophogenicity of the preparation, a secondary effect of topical corticosteroids related with their potency. Results confirm that L are an effective topical drug delivery system.· P stabilized o/w emulsions with 2% w/w BO were obtained with the M110S. Emulsions, hydrogels and lyophilized samples show a good stability. Vesicle composition (L and droplets) are dependent on the initial BO/P ratio.

Bio-nanotechnology is the use of biomolecules to control both the structure and property of nanomaterials. No biomolecule has been employed more often than DNA as exemplified in the numerous demonstrations of DNA-directed assembly of nanomaterials. DNA has been used to covalently functionalize and assemble soft nanoparticles (e.g. liposomes) and hard nanoparticles (e.g. gold and silica nanoparticles) into a variety of hierarchical nanostructures. The majority of previous work however has focused on the latter, i.e., the assembly of “hard” nanoparticles such as gold nanoparticles (AuNPs) as oppose to the assembly of soft materials. The primary focus of this thesis is to add to the growing field of DNA-directed assembly of soft materials owing to the promise of such materials in a variety of analytical and biomedical applications. The first class of soft materials considered are liposomes which interestingly can be deformed by relatively weak intermolecular forces. In addition, DNA anchored to its surface can readily diffuse laterally within the lipid bilayer while DNA attached to inorganic nanoparticles remain fixed in position. We systematically consider the effect of varying the liposome structure, size, charge, and fluidity on liposome assemblies, in chapter 2. In addition, the interesting properties of liposomes are highlighted by a side-by-side comparison to DNA-functionalized gold nanoparticles, offering fundamental insights into DNA-directed assembly. Furthermore, hybrid DNA-directed assemblies composed of both AuNPs and liposomes are described in Chapter 3. In particular, the photothermal effects of such DNA-coupled liposome and AuNP assemblies were modulated by controlling the distance between liposome and AuNP allowing such systems to have potential application in drug-delivery. In chapter 4, the utility of liposomes is demonstrated as we exploit the fluidity of its diffuse bilayer with split aptamer functionalization for the rapid and selective detection of metabolites. The second class of soft material of interest in this thesis are hydrogels, which are cross-linked hydrophilic polymers. Because hydrogels are swollen in water, they can be used to immobilize biomolecules such as DNA for a myriad of applications. In chapter 5, the preparation and characterization of DNA-functionalized polyacrylamide hydrogels are presented. The use of such a DNA-modified hydrogel for the simultaneous detection and removal of mercury from water is subsequently demonstrated.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) have been shown to stimulate the innate immune system against a variety of bacterial, viral, and protozoan infections in a variety of vertebrate species. The objectives of this study were to investigate the immunostimulatory effect of CpG-ODN against Salmonella Typhimurium infection and the formulation and delivery of CpG-ODN by the in ovo route. Day-old broiler chicks or embryonated eggs (day 18th of incubation) received either 50 g of CpG-ODN, 50 g of non-CpG-ODN, or saline. At day four-post hatch, all birds were subcutaneously inoculated by Salmonella Typhimurium. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for ten days following challenge. The survival rate of the birds that received CpG-ODN via in ovo or in vivo treatments was significantly higher than the control group. Salmonella Typhimurium level in the peripheral blood and pathology were significantly lower (p < 0.001) in CpG-ODN group compared to the control group. In order to investigate the effect of formulation of CpG-ODN, embryonated eggs (day 18th of incubation) were inoculated with either 50 g of CpG-ODN alone or CpG-ODN formulated with polyphosphazene, liposome, or Emulsigen®. Four days after administration of CpG-ODN formulations, the birds were challenged with E. coli by subcutaneous injection. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for seven days following challenge. The birds that received either CpG-ODN or CpG-ODN formulated with polyphosphazene had significantly higher survival rates (30 and 60%) compared to the birds in groups receiving either non-CpG-ODN or saline. Bacterial loads in the air sacs were lower in groups treated with formulated CpG-ODN compared to the CpG-ODN alone or control groups. However, formulation of CpG-ODN with liposomes or Emulsigen® did not increase the immunoprotective effect against E. coli infection. We showed that treatment with CpG-ODN protects neonatal chickens against an intracellular bacterial infection and that co-treatment of CpG-ODN with polyphosphazene enhances the immunoprotective effect of CpG-ODN.

<p>Liposomes are excellent chemotherapy drug delivery agents, on the cutting edge of cancer treatment technology. Since liposomes are already used to deploy cancer drugs in patients, imaging capacity would make them dual-purpose "theranostic" vesicles. Intermolecular double quantum coherence (iDQC) MRI is uniquely suited to this application, as its contrast does not require any additional chemicals. Adding contrast agents to liposomes can be time-consuming, add to toxicity, interfere with membrane function, or adversely affect drug loading. Furthermore, iDQC contrast measures diffusion and thus directly depends on membrane permeability and related properties. In this set of experiments, it has been shown that iDQC signal from intra-liposomal water can be distinguished from that of bulk water, and that the T2 dynamics of intra-liposomal water are predictable and dependent on the percent of water encapsulated. These techniques to distinguish between water molecules based on their current physical circumstances lead to many novel possibilities in MRI, as nearly all the signal in conventional MRI is from water protons. Based on the signal to noise ratio in the aforementioned iDQC experiments, we predict that iDQC contrast from liposomes will be visible in vivo, and propose to prove this in a murine model. By examining intra-liposomal water, iDQC can be used to improve chemotherapy delivery via real time monitoring of liposome location and drug release.</p> / Thesis

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Not available / text

Ο σκοπός της παρούσης εργασίας είναι να εξετάσουμε την κινητική απελευθέρωσης υδρόφιλων και λιπόφιλων μορίων που εγκλωβίζονται σε λιποσώματα, όταν τα λιποσώματα διασπείρονται σε συστήματα φορέων- υδρογελών. Στην παρούσα εργασία μελετάται η απελευθέρωση της υδατοδιαλυτής ουσίας καλσεΐνης και της λιποδιαλυτής γκριζεοφλουβίνη τόσο από σταθερά λιποσώματα (DSPC:Chol) όσο και από ασταθή (PC) διεσπαρμένα σε φορείς υδρογελών. / Release of calcein and griseofulvin (GRF) from control (gels in which solutes are dissolved in) and liposomal gels was studied using agarose-assisted immobilization as a technique to separate gels from drug-receptor compartments.Results show that calcein release from liposomal gels is slower compared to control gels, and can be further retarded by using rigid-membrane liposomes (faster release from PC-liposome compared to DSPC/Chol-liposome gels).Additionally, calcein release is not affected by the lipid amount loaded (in the range from 2 to 8 mg/ml), therefore solute loading can be controlled according to needs. Oppositely, GRF release from liposomal gels is determined by drug loading. At high drug loading levels (compared to GRF aqueous solubility), GRF is released with constant rate from liposomal gels irrespective of liposome type (PC or DSPC/Chol). Thereby, for amphiphilic/lipophilic drugs, drug properties (solubility, log P) determine the system behavior. Calcein and GRF release from control carbopol gels is faster compared to HEC and mixture gels. The same is true for calcein in liposomal gels. Carbopol gel rheological properties were found to be significantly different (compared to the other gels), implying that these characteristics are important for drug diffusion from gels.

Είναι γνωστό ότι τα λιποσώματα παρέχουν πολλά πλεονεκτήματα για τη χορήγηση και /ή τη στόχευση φαρμάκων (Lasic 1993, Gregoriadis 1988). Κατά τη χορήγηση λιποσωμικών φαρμακομορφών για τοπική (topical) εφαρμογή ή εφαρμογή σε επιθήλια (mucosal) (Rollan 1993, Schreier and Bouwstra 1994) είναι απαραίτητο οι ρεολογικές και βλεννοσυγκολητικές ιδιότητες των λιποσωμικών διασπορών να ρυθμίζονται ανάλογα με την επιδιωκόμενη οδό χορήγησης. Αυτό μπορεί εύκολα να επιτευχθεί με τη προσθήκη παραγόντων αύξησης ιξώδους (gelling agents) στις λιποσωμικές διασπορές. Συνεπώς προκύπτουν σύνθετες φαρμακοτεχνικές μορφές που από δομικής σύστασης είναι διασπορές λιποσωμικών μορφών φάρμακων σε συστήματα γελών (drug-in-liposome-in-gel). Ανάλογα με το βαθμό συγκράτησης του φαρμάκου στα λιποσώματα μετά τη διασπορά των λιποσωμάτων στην επιθυμητή φαρμακοτεχνική μορφή, μπορεί να τροποποιηθεί και ο ρυθμός απελευθέρωσης του φαρμάκου από τέτοια συστήματα. Αυτό συνδέεται σε μεγάλο βαθμό κυρίως με δύο ομάδες παραγόντων: • Πρώτον με τη σταθερότητα των λιποσωμάτων (ακεραιότητα μεμβράνης και μηχανική σταθερότητα) κατά τη διασπορά σε ημι-στερεές φαρμακοτεχνικές μορφές, κάτι το οποίο συνδέεται τόσο με την σκληρότητα (rigidity) της μεμβράνης του λιποσωμικού φορέα όσο και με τις φυσικές ιδιότητες του ημι-στερεού συστήματος (ιξώδες και ρεολογικές ιδιότητες). • Δεύτερον, με τις φυσικοχημικές ιδιότητες του προς μορφοποίηση φάρμακου. Όσο πιο λιπόφιλο είναι ένα φάρμακο και όσο μικρότερη η διαλυτότητα του στο νερό, τόσο μεγαλύτερος θα είναι λογικά ο χρόνος συγκράτησης του στις λιπιδικές διπλοστοιβάδες των λιποσωμάτων συγκριτικά με αμφίφιλα φάρμακα που έχουν σημαντικά υψηλότερη διαλυτότητα στο νερό. Επειδή σε ανάλογα σύνθετα (φάρμακο-σε-λιπόσωμα-σε-γέλες) συστήματα χορήγησης είναι πιθανόν να πρέπει να συνυπάρχουν εκτός από τον παράγοντα αύξησης του ιξώδους και άλλα έκδοχα, κυρίως παράγοντες που αυξάνουν τη διαλυτότητα διάφορων ουσιών ή επιφανειοδραστικά, ο σκοπός της παρούσας μελέτης ήταν η εξέταση της επίδρασης τέτοιων εκδοχών κατά την προσθήκη τους στα παραπάνω συστήματα. Πιο συγκεκριμένα μελετήθηκε η επίδραση των εξής εκδόχων: Transcutol, Propylene-glycol, Cremophor και Labrafac στη σταθερότητα λιποσωμικών μορφών φαρμάκων και στο ρυθμό απελευθέρωσης υδρόφιλων και λιπόφιλων μορίων από συστήματα διασποράς λιποσωμικών μορφών τέτοιων ουσιών σε γέλες (drug-in-liposome-in-gel). Τα έκδοχα αυτά μόνα ή σε συνδυασμό μεταξύ τους καθως και σε συνδυασμό με άλλους φορείς προσφέρονται για την παρασκευή κατάλληλων φαρμακοτεχνικών μορφών που αποβλέπουν στην αύξηση διαλυτότητας, απορρόφησης και τελικά της βιοδιαθεσιμότητας δυσδιάλυτων φαρμάκων Για την εκτίμηση του βαθμού επίδρασης των πιο πάνω αναφερθέντων εκδοχών στη σταθερότητα λιποσωμικών μορφών φαρμάκων και στο ρυθμό απελευθέρωσης τόσο των υδρόφιλων όσο και λιπόφιλων φάρμακων από συστήματα διασποράς λιποσωμικών μορφών τους σε γέλες (drug-in-liposome-in-gel). χρησιμοποιήθηκε ως μοντέλο υδρόφιλων μορίων η καλσεΐνη ενώ ως μοντέλο υδρόφοβων ουσιών η γκριζεοφουλβίνη. Εδώ ως παράγοντες αύξησης του ιξώδους για την παρασκευή των σύνθετων φαρμακευτικών μορφών, φάρμακο-σε-λιπόσωμα-σε-γέλες χρησιμοποιήθηκαν: (Ι) το πολυμερές του ακρυλικού οξέος, carbopol 974PNF, (ΙΙ) το πολυμερές υδροξυ-αιθυλ-κυτταρίνης (HEC-Hydroxyethylcellulose) καθως και (ΙΙΙ) μίγμα των δυο πολυμερών. Γενικά από τη μελέτη αυτή συμπεραίνουμε ότι: Η επίδραση των προς εξέταση παραγόντων στη σταθερότητα των λιποσωμάτων εξαρτάται από: τη λιπιδική σύσταση, την προσθήκη χοληστερόλης κατά την παρασκευή των λιποσωμάτων, τη λιποφιλικότητα του εκδόχου και τη συγκέντρωσή του. Σχετικά με τη σταθερότητα των λιποσωμάτων παρουσία των εκδόχων που μελετήθηκαν: 1. Tα PC λιποσώματα σπάνε πολύ εύκολα με και χωρίς την παρουσία των εκδοχών στο φορέα υδρογέλης. 2. Τα πιο ανθεκτικά λιποσώματα DSPC/Chol 1:1 φαίνεται να έχουν μεγαλύτερη σταθερότητα. Όμως και στις δυο περιπτώσεις (PC και DSPC/Chol) η σταθερότητα των λιποσωμάτων στους παράγοντες αυτούς μειώνεται ακολουθώντας την εξής σειρά: Transcutol ≈ Propylene-glycol < Cremophor < Labrafac. 3. To Labrafac διαταράσσει στο μεγαλύτερο βαθμό τις λιπιδικές διπλοστοιβάδες προκαλώντας τη μεγαλύτερη διαρροή των εγκλωβισμένων μορίων προκαλώντας σημαντικές δομικές μεταβολές στα λιποσώματα. 4. H χοληστερόλη αυξάνει την σταθερότητα των λιποσωμάτων κυρίως παρουσία των εκδοχών με μια σχετική ασθενή δράση αλλά όχι σημαντικά παρουσία του εκδόχου Labrafac. Σχετικά με τη κινητική απελευθέρωσης ουσιών από λιποσωμικές τους μορφές που διασπείρονται σε γέλες παρουσία των εκδόχων που μελετήθηκαν: 1. Ένα σημαντικό εύρημα είναι ότι υπάρχει μεγάλη διαφορά στη συμπεριφορά απελευθέρωσης λιπόφιλων και υδρόφιλων μορίων από λιποσωμικές μορφές τους όταν τα τελευταία διασπείρονται σε συστήματα υδρογέλων παρουσία και απουσία των εκδόχων καθως και στη μεταξύ τους απελευθέρωση. 2. Σε γενικές γραμμές η απελευθέρωση των εγκλωβισμένων στα λιποσώματα ουσιών, όταν αυτά διασπείρονται στις γέλες που περιέχουν έκδοχα, φαίνεται να συνδέεται-επηρεάζεται από τη σταθερότητα των λιποσωμάτων παρουσία αυτών των εκδόχων, όπως ευρέθηκε στο πρώτο μέρος της μελέτης. Η κινητική απελευθέρωσης λιπόφιλων φαρμάκων από σύνθετα συστήματα όπως αυτά που μελετήθηκαν μπορεί να επιβραδύνεται σημαντικά όταν μέσα στις μορφές προστίθενται έκδοχα με υψηλή λιπόφιλα (Labrafac- γκριζεοφουλβίνη) (πιθανή κατανομή της γκριζεοφουλβίνης σε λιπόφιλες περιοχές). Κλείνοντας επισημαίνουμε ότι η ανακάλυψη νέων συστημάτων χορήγησης φαρμάκων αποτελεί σήμερα τη μεγαλύτερη πρόκληση για τους επιστήμονες. Οι πιο πάνω φορείς συγκαταλέγονται ανάμεσα στα πιο σπουδαία συστήματα χορήγησης φαρμάκων όχι μόνο λόγω συμβατότητας τους ως προς το ανθρώπινο οργανισμό αλλά και εξαιτίας της μεγάλης δυνατότητας εφαρμογών που προσφέρουν. Απώτερος σκοπός αυτής της μελέτης είναι η καταχώρηση στη βιβλιογραφία νέων στοιχείων σχετικά με την επίδραση αυτών των εκδοχών και συνεπώς η διευκόλυνση κατά την επιλογή των καταλληλότερων συνδυασμών φορέων-εκδοχών στο σχεδιασμό επιθυμητών φαρμακοτεχνικών μορφών. / It is well known that liposomes offer many advantages for the delivery and/or targeting of drugs (Lasic 1993, Gregoriadis 1988). When mucosal or topical delivery of liposomal formulations is considered (Rollan 1993, Schreier and Bouwstra, 1994), it is essential that rheological and/or mucoadhesive properties of the liposome dispersions are adjusted accordingly, depending on the intended route of administration. This can be easily dome by adding gelling agents in the liposomal dispersions. Thereby, eventually a drug-in-liposome-in gel complex formulation is developed. Depending on the retention of the drug in the liposomes after the liposomes are dispersed in the preferred formulation, the release rate of the drug may be modified. This is highly related with two major groups of factors; • First the stability of the liposomes (membrane integrity and mechanical stability) during dispersion in the semi-solid formulation that may be related to the vesicle-membrane rigidity as well as the semisolid system physical properties (viscosity and rheological properties). • Second, the physicochemical properties of the drug formulated. A more lipophilic drug with low aqueous solubility should be logically retained for longer time periods in liposome lipid bilayers when compared with amphiphillic drugs with considerable high aqueous solubility (that will be the driving force to move drug molecules to partition in the aqueous environments until saturation occurs). In some cases, in addition to the gelling agents it is essential to have also other excipients in the complex drug-in-liposome-in-gel systems, as solubilizers, surfactants, co-surfactants et.c. Herein, we evaluate the effect of such excipients on the release of drugs from complex systems. We chose to use trancutol, propylene glycol, cremophor and labrafac-hydro, that are commonly used excipients in pharmaceutical formulations. In order to evaluate the extent to which these compounds can affect the release rate of drugs from drug-in-liposome-in-gel systems we followed the release of two model compounds, one hydrophilic dye (calcein) and one lipophilic drug (griseofulvin). The effect of the rigidity of liposomal membranes was evaluated by testing two different liposome lipid compositions for each case of encapsulated compounds, one that is known to be what is called “leaky”, which is the plain PC (egg lecithin) composition, and one which is very rigid, which is the DSPC/Chol (1:1 mol/mol) composition. Additionally, we also evaluated the effect of the gel composition and characteristics, by studying the release of both drugs from liposomes that have been dispersed in systems with different properties. For this we used gels composed of the acrylic acid based polymer Carbopol 974, which is a substance of many commercially available semisolid formulations, at two different concentrations (same rheological properties and different viscosity) as well as a cellulose based gel, composed of hydroxyethyl-cellulose (different rheological properties). Additionally we evaluated also a mixture of the two polymers, which has been proposed as a formulation base with several advantages, for vaginal delivery of drugs. In addition, we studied the stability of liposomes during incubation in the presence of the plain excipients (in 2 different concentrations, 10% and 25%) in order to see if this can be correlated on the effect of the same agents on drug release from the complex systems. The general conclusion extracted from the experimental results of this study is that the effect of the excipients on liposome stability depend on (i) the lipid composition of the liposomal membranes, (ii) the inclusion of cholesterol in the liposomes or not, (iii) the lipophilicity of the excipient and (iv) the concentration of the excipient. More analytically: In respect to liposome stability: 1. PC liposomes are easily disrupted with or without the presence of the excipients in the gels. 2. The very rigid DSPC/Chol (1:1 mol/mol) liposomes demonstrate higher stability. 3. In both cases the effect of the excipients follows the sequence: Transcutol ≈ Propylene-glycol < Cremophor < Labrafac. 4. Labrafac has the highest effect on liposome stability it practically dissolves liposomal membranes. 5. Cholesterol inclusion in liposomal membranes results in increased stability, however even the most stable DSPC/Chol (1:1) liposomes are very unstable in presence of Labrafac. In respect to the release of liposomal drugs from the complex systems in presence of the excipients studied: 3. A significant finding is that there is a difference in the release pattern and rate between lipophilic and hydrophilic liposome-encapsulated molecules, in the presence of excipient or not (control studies).. 4. In general, there seems to be a correlation between the stability of liposomes in presence of excipients and the effect that the specific excipient has on the release kinetics of liposome encapsulated molecules from complex drug-in-liposome-in-gel systems. 5. The release of lipophilic molecules (that are encapsulated in the lipospmes) from complex systems can be substantially sustained when lipophilic excipients are added in the gel (as is the case of griseofulvin and labrafac). Concluding, we stress the fact that the invention of new drug delivery systems with better performance is a great challenge for scientists involved in pharmaceutics. The systems studied here are offering many advantages due to their biocompatible nature and the extended number of applications they may have. The final scope of this study is the entry of new data about the effect of excipients on the properties of complex drug delivery systems. As the availability similar data in the related literature increases, it will become more easy to make the best selection of excipients during the development of better formulations for existing of new drugs.