Biomedical subjects

D J Crommelin

Publications and source records attributed to D J Crommelin.

At least 19 recordsLinked to original sources

PubMed2001

Targeting influenza virosomes to ovarian carcinoma cells.

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) have attracted attention as delivery vesicles for cytosolic drug delivery as they possess membrane fusion activity. Here, we show that influenza virosomes can be targeted towards ovarian carcinoma cells (OVCAR-3) with preservation of fusion activity. This was achieved by incorporating poly(ethylene glycol) (PEG)-derivatized lipids into the virosome membrane. This PEG layer serves as shield to prevent interaction of HA with ubiquitous sialic acid residues and as spatial anchor for antibody attachment. Coupling of Fab' fragments of mAb 323/A3 (anti-epithelial glycoprotein-2) to the distal ends of PEG lipids resulted in specific binding of virosomes to OVCAR-3 cells. These antibody-redirected virosomes fused with membranes of OVCAR-3 cells in a pH-dependent fashion.

Cell Membrane↗

PubMed2001

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.

Amides↗

PubMed2001

Liposomes as sustained release system for human interferon-gamma: biopharmaceutical aspects.

Interferon-gamma (IFNgamma) has proven to be a promising adjuvant in vaccines against cancer and infectious diseases. However, due to its rapid biodegradation and clearance, its efficacy is severely reduced. Liposomal association might prolong the residence time of IFNgamma, but no efforts have been made to optimize the biopharmaceutical characteristics of liposomal IFNgamma for its application in therapy or as vaccine immunoadjuvant. In the present study, various liposomal formulations of recombinant human IFNgamma (hIFNgamma), differing in lipid composition, were prepared via the film hydration method and characterized in vitro regarding association efficiency and bioactivity, and in vivo regarding cytokine release kinetics after subcutaneous (s.c.) administration into mice. Human IFNgamma can be formulated in large, multilamellar liposomes with high association efficiency (>80%) and preservation of bioactivity. A critical parameter is the inclusion of negatively charged phospholipids to obtain a high liposome association efficiency, which is dominated by electrostatic interactions. The fraction of externally adsorbed protein compared to the total associated protein can be minimized from 74+/-9% to 8+/-3% by increasing the ionic strength of the dispersion medium. After injection of free (125)I-hIFNgamma, the radiolabel was detectable up to 48 h at the injection site. Liposomal encapsulation of (125)I-hIFNgamma increased the local area under the curve 4-fold, and the presence of the radiolabeled hIFNgamma at the injection site was prolonged to 7 days. The release kinetics and overall residence time of the cytokine at the s.c. administration site was influenced by depletion of the externally adsorbed IFNgamma, reducing the initial burst release. Increasing the rigidity of the liposome bilayer also resulted in a more pronounced reduction of the burst release and a 19-fold increase in the residence time of the protein at the s.c. administration site, compared to the free cytokine. As adjuvanticity of liposomal IFNgamma may strongly depend on the release kinetics of cytokines in vivo, the findings in this paper may contribute to a rational design of liposomal-cytokine adjuvants in vaccines against cancer and infectious diseases.

Adjuvants, Immunologic↗

PubMed2001

Degradable dextran microspheres for the controlled release of liposomes.

A novel delivery concept based on the encapsulation of liposomes in biodegradable dextran microspheres was developed. The microspheres were prepared using a two-phase system, consisting of water/poly(ethylene glycol), and water/methacrylated dextran. Liposomes were encapsulated almost quantitatively and in their intact form, and were released with full preservation of their integrity. The effects of microsphere water content, degree of methacrylate substitution, and type of dextran derivative used on the release rate were investigated. The release of the liposomes from the dextran microspheres was fully controlled by the degradation rate of the spheres. This resulted, after a lag time, in a pulsed release of the liposomes from relatively rapidly degrading microspheres. On the other hand, slower degrading microspheres resulted in sustained release of liposomes over 100 days. The degradation rate of the dextran microspheres, in turn, depended on the water content, the degree of methacrylate substitution, and type of hydrolytically sensitive spacer present in the cross-links.

Dextrans↗

PubMed2001

The fate of poly(2-dimethyl amino ethyl)methacrylate-based polyplexes after intravenous administration.

Poly(2-dimethyl amino ethyl) methacrylate (pDMAEMA) cationic polymers have been shown to be efficient vectors for gene delivery in vitro. This contribution deals with the in vivo properties of polyplexes based on this polymer. In mice, pDMAEMA/[32P]-pLuc complexes distributed primarily to the lungs. The gene expression profile matched the biodistribution profile. In vitro turbidity experiments in serum showed severe aggregation upon addition of cationic polyplexes, pointing out the involvement of aggregates in the dominant lung uptake of the positively charged polyplexes. Incubations of polyplexes with albumin yielded a decline of the zeta potential of the complexes to negative values, making an electrostatic mechanism in the dominant lung uptake less likely. Hemagglutination experiments showed that the polyplexes induce the formation of extremely large structures when incubated with washed erythrocytes. Altogether, the present data indicate that aggregate formation and trapping of the formed aggregates in the lung capillary bed is probably responsible for the dominant lung uptake and transfection. Poly(ethylene)glycol (PEG) of the polymeric structures prevented the increase in the observed turbidity in serum seen with polyplexes and was also able to reduce interactions with erythrocytes. Currently, the in vivo fate of the PEGylated polyplexes is under investigation.

Animals↗

PubMed2001

The influence of the incorporation of cholesterol and water on the particle size, bilayer thickness, melting behavior, and relative sucrose ester composition of reversed vesicles.

The influence of the incorporation of cholesterol and water on the particle size, bilayer thickness, melting behavior, and relative sucrose ester composition of reversed vesicles was studied. Reversed vesicles (RVs) were prepared of sucrose ester in silicon oil by sonication. The RVs were characterized by polarized light microscopy, laser diffraction, high-performance liquid chromatography, small angle X-ray scattering (SAXS), and differential scanning calorimetry. The particle size distributions of the studied dispersions were bimodal with peaks at 5 and 0.4 microm. There was no significant difference in the sucrose ester composition of these two size categories of RVs. The incorporation of cholesterol and water had no effect on the size distribution of the RVs. The SAXS results showed that the RVs prepared without cholesterol and water consisted of bilayers with fully interdigitated alkyl chains. The incorporation of high concentrations of cholesterol caused a phase separation within the bilayers. The incorporation of water also resulted in a phase separation within the bilayers but at a lower cholesterol concentration. The presence of two different size classes of RVs in one RVs dispersion and the phase separation within the bilayers of certain compositions can have consequences for the application of RVs.

Cholesterol↗

PubMed2001

Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells.

A nonviral gene delivery vector has been developed in our laboratory based on the cationic polymer, poly(2-(dimethylethylamino)ethyl methacrylate) (p(DMAEMA)). p(DMAEMA)-based polyplexes have been successfully used for the transfection of OVCAR-3 cells in vitro. However, these polyplexes were unable to transfect OVCAR-3 cells growing in the peritoneal cavity of nude mice after intraperitoneal administration, which could be ascribed to inactivation by components (including hyaluronic acid) present in the tumor ascitic fluid. The present work aimed at (a) protecting p(DMAEMA)-based polyplexes against destabilization or inactivation by polyanions such as hyaluronic acid present in tumor ascitic fluid and (b) enhancing cellular uptake of the protected p(DMAEMA)-based polyplexes by targeting with antibody Fab' fragments. To fulfill these requirements, we have developed a detergent removal method to coat polyplexes with anionic lipids. With this method, spherical particles of approximately 125 nm, which were protected from destabilization by polyanions, were obtained. More importantly, the transfection efficiency of lipopolyplexes was unaffected in the presence of hyaluronic acid, indicating that lipid coating of polyplexes protects against destabilization by hyaluronic acid. By conjugating antibody Fab' fragments directed against the epithelial glycoprotein-2 to the lipidic surface of these lipopolyplexes, target cell-specific transfection of OVCAR-3 cells could be obtained in vitro.

Animals↗

PubMed2000

Sterilization of poly(dimethylamino) ethyl methacrylate-based gene transfer complexes.

Parental administration of polyplex formulations for gene therapy or genetic vaccination requires sterile preparations. The possibilities and limitations of autoclaving, filtration and a combination of both methods for sterilization of poly(2-(dimethylamino) ethyl methacrylate) (pDMAEMA) based gene transfer complexes were assessed. Agarose gel electrophoresis and circular dichroism spectroscopy showed that sterile filtration of polyplexes did not change the topology and integrity of the DNA. The transfection potential was fully retained in COS-7 and OVCAR-3 cells, although the concentration of DNA was slightly decreased by the filtration process. Pre-coating of the filter with polyplexes reduced the material loss. In contrast, autoclaving dramatically affected physical characteristics of polyplexes, resulting in complete loss of transfection potential. Sterile filtration or autoclaving of polymer alone did not result in material loss, or in decreased transfection potential after complexation with plasmid DNA. 'Naked' DNA could easily be sterilized by filtration as well. In conclusion, sterilization of complexes between pDMAEMA-based cationic polymeric gene transfer agents and DNA plasmid is feasible by filtration. Depending on the filter type used, the filtered volume should be high enough, to prevent substantial material loss. Separate sterilization of the polymer by autoclaving or filtration and DNA by filtration offers a good alternative to filtration of formed polyplexes.

Animals↗

PubMed2000

The effect of a water/organic solvent interface on the structural stability of lysozyme.

The effect of emulsification of lysozyme solutions with methylene chloride on protein recovery and structural integrity was investigated. Total lysozyme recovery in the aqueous phase was found to be concentration dependent, and ranged between 65 and 80%. The unrecovered lysozyme was observed at the interface as a white precipitate. No structural changes of the soluble lysozyme were observed by enzymatic activity assay, size-exclusion chromatography (SEC), gel electrophoresis (SDS-PAGE), and circular dichroism (CD). The lyophilized precipitated protein was analyzed by FTIR, and evidence of intermolecular beta-sheet formation was found. In addition, the precipitate was analyzed after redissolution in 1 M guanidine hydrochloride by enzymatic activity assay, CD, SDS-PAGE, and SEC. No differences with control lysozyme samples or samples in aqueous buffer solutions were observed. This indicates that lysozyme precipitates as non-covalent aggregates upon emulsification, and these precipitates can refold into their native state in 1 M guanidine hydrochloride. Protein recovery could not be improved by the addition of sucrose, Tween 20, or Tween 80. Excipients competing for the water/organic solvent interface, such as BSA and partially hydrolyzed polyvinylalcohol (PVA) significantly improved lysozyme recovery to >95%. Emulsions which contained poly(lactic-co-glycolic acid) (PLGA) in the organic phase gave irreproducible protein recovery. Here also, partially hydrolyzed PVA significantly increased lysozyme recovery. Thus, we found that emulsification of lysozyme-containing aqueous solutions with methylene chloride causes incomplete protein recovery and non-covalent aggregation of lysozyme. These aggregates are also encapsulated in controlled drug delivery systems which are prepared using a water-in-oil emulsification procedure. The use of surface-active additives, such as partially hydrolyzed PVA significantly reduces lysozyme aggregation, and can be used to prevent encapsulation of inactive and potentially immunogenic protein species.

Anti-Infective Agents↗

PubMed2000

Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques.

Lysozyme distribution and conformation in poly(lactic-co-glycolic acid)(PLGA) microspheres was determined using various infrared spectroscopic techniques. Infrared microscopy and confocal laser scanning microscopy indicated that the protein was homogeneously distributed inside the microspheres in small cavities resulting from the water-in-oil emulsification step. Part of the protein was observed at or near the cavity walls, while the rest was located within these cavities. Attenuated total reflectance (ATR) and photoacoustic spectroscopy (PAS) also showed that there is hardly any protein at the surface of the microspheres. Since this microsphere formulation gave a large burst release (ca. 50%), this burst release can not be caused by protein at the surface of the particles. Probably, the protein is rapidly released through pores in the PLGA matrix. Conformational analysis of lysozyme in the PLGA microspheres by KBr pellet transmission suffered from band shape distortion and baseline slope. Despite incomplete subtraction of the PLGA background, a characteristic band of non-covalent aggregates at 1625 cm(-1) was observed in the second derivative spectrum of the protein Amide I region. The other Fourier-transform infrared (FTIR) methods yielded similar results, indicating that the sample preparation procedure did not introduce artifacts. The observed aggregation signal may correspond to the protein adsorbed to the cavity walls inside the microspheres.

Anti-Infective Agents↗

PubMed2000

Copolymers of 2-(dimethylamino)ethyl methacrylate with ethoxytriethylene glycol methacrylate or N-vinyl-pyrrolidone as gene transfer agents.

Random copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethoxytriethylene glycol methacrylate (triEGMA) or N-vinylpyrrolidone (NVP) of different molecular weights and compositions were synthesized, characterized and evaluated as polymeric transfectants in vitro. All synthesized copolymers (comonomer fraction up to 66 mol%) were able to bind to DNA, yielding polymer-plasmid complexes (polyplexes). However, the polymer-plasmid ratio at which small complexes (size 0.2-0.3 microm) were formed, increased with increasing mole fraction of the comonomer. zeta-Potential measurements revealed that the polymer-plasmid ratio where charge neutralization of DNA occurred, increased with increasing mole fraction of triEGMA. The cytotoxicity of the copolymers, either complexed with DNA or in the free form, decreased with increasing mole fraction of both comonomers (triEGMA and NVP). This reduction was even more than what could be expected based on the DMAEMA mole fraction in the copolymer. The copolymers with a molecular weight up to 170¿ omitted¿000 had the same transfection capability as a homopolymer of comparable molecular weight. However, higher molecular weight copolymers showed a reduced transfection capability compared to the homopolymer, which was ascribed to the reduced capability to condense the size of plasmid. Transfection efficiency of polyplexes composed of copolymers with a low triEGMA content increased with increasing molecular weight. Although the copolymers with 50 mol% triEGMA were also better transfectants than the homopolymer, the transfection efficiency did not increase further with increasing molecular weight. Interestingly, NVP-DMAEMA copolymers synthesized by polymerization to high conversion showed both excellent DNA binding and condensing characteristics (polyplex size <0.3 microm) and transfection capabilities. This is ascribed to a synergistic effect of DMAEMA-rich copolymers and NVP-rich copolymers present in this system on the complex formation with plasmid DNA.

Chromatography, High Pressure Liquid↗

PubMed2000

Conformational stability of human interferon-gamma on association with and dissociation from liposomes.

The integrity of a therapeutic protein has to be safeguarded when formulated in delivery systems such as liposomes. In this study, we investigated the conformational stability of recombinant human interferon gamma (hIFNgamma) on association with and after dissociation from liposomal bilayers using circular dichroism (CD) and steady-state fluorescence spectroscopy as well as time-resolved fluorescence methodology. We used hIFNgamma adsorption to and desorption from empty liposomes as a model for hIFNgamma-containing liposomes prepared via the film hydration method. CD studies indicated that no changes in the secondary and tertiary protein structure occur during and after interaction of hIFNgamma with the liposomes. Steady-state fluorescence emission spectra of untreated and liposome-desorbed hIFNgamma revealed that the environment of the sole Trp residue was not affected by the adsorption/desorption process. The Trp-36 residue remained fully quenchable by acrylamide after desorption of hIFNgamma from the liposomes. Time-resolved fluorescence studies were conducted to probe the local environment and the mobility of Trp-36 before, during, and after interaction of hIFNgamma with the liposomal membrane. Differences in rotational correlation time between free and liposomal hIFNgamma were attributed to immobilization of the protein on adsorption to the liposome bilayer. Disparities were detected between the average lifetimes of liposome-adsorbed hIFNgamma and hIFNgamma-liposomes, indicating that subtle changes in the Trp-36 environment took place during preparation of the liposomes via the film hydration method compared with the adsorption of hIFNgamma to the liposome surface. The results of this study indicate that association of hIFNgamma with negatively charged liposomes results in minimal changes in the secondary and tertiary structure of the protein. We conclude that all techniques used point to a full retention or restoration of the protein conformation after desorption from the liposomes.

Acrylamide↗

PubMed2000

Liposomes containing interferon-gamma as adjuvant in tumor cell vaccines.

PURPOSE: Liposomal systems may be useful as a cytokine supplement in tumor cell vaccines by providing a cytokine reservoir at the antigen presentation site. Here, we examined the effect of liposome incorporation of mIFNgamma on its potency as adjuvant in an established tumor cell vaccination protocol in the murine B16 melanoma model. Adjuvanticity of the mIFNgamma-liposomes was compared to that achieved by mIFNgamma-gene transfection of the B16 tumor cells. Furthermore, we studied whether liposomal incorporation of mIFNgamma indeed increases the residence time of the cytokine at the vaccination site. METHODS: C57B1/6 mice were immunized with i) irradiated IFNgamma-gene transfected B16 melanoma cells or ii) irradiated wild type B16 cells supplemented with (liposomal) mIFNgamma, followed by a challenge with viable B16 cells. The residence time of the (liposomal) cytokine at the subcutaneous (s.c.) vaccination site was monitored using radiolabeled mIFNgamma and liposomes. RESULTS: Immunization with irradiated tumor cells admixed with liposomal mIFNgamma generated comparable protection against B16 challenge as immunization with mIFNgamma-gene modified tumor cells. Irradiated tumor cells admixed with soluble mIFNgamma did not generate any protective responses. Radiolabeling studies indicated that free mIFNgamma rapidly cleared from the s.c. injection site. Association of [125I]-mIFNgamma with liposomes increased the local residence time substantially: liposomal association of mIFNgamma resulted in a prolonged local residence time of the cytokine as reflected by a 4-fold increase of the area under the curve. The amount of released cytokine in the optimal dose range corresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for mice immunized with irradiated cells supplemented with mIFNgamma-liposomes compared to untreated control animals. CONCLUSIONS: Prolonged presence of mIFNgamma at the site of antigen presentation is crucial for the generation of systemic immune responses in the B16 melanoma model. These studies show that liposomal encapsulation of cytokines is an attractive strategy for paracrine cytokine delivery in tumor vaccine development.

Adjuvants, Immunologic↗

PubMed2000

Controlled release of liposomes from biodegradable dextran microspheres: a novel delivery concept.

PURPOSE: To design liposome-loaded microspheres, which release the liposomes in a time-controlled manner and in intact form. METHODS: Liposomes were encapsulated in biodegradable dextran-based microspheres, which were prepared using an aqueous two phase system consisting of poly(ethylene glycol) and methacrylated dextran. The effects of liposome size and membrane fluidity, microsphere water content, degree of methacrylate substitution, and type of dextran derivative used, on encapsulation efficiency, release, and integrity of the liposomes were investigated. RESULTS: Liposomes were entrapped in dextran-based microspheres quantitatively and with full preservation of their integrity. Liposomes with a low, as well as with a high membrane fluidity, were released from the microspheres in their intact form and with preservation of their size. Release kinetics depended only on the degradation rate of the microspheres. For rapidly degrading systems, pulsed release was observed and the time after which the pulse occurred (from 5 until 25 days) could be tailored by the gel characteristics such as initial water content, degree of methacrylate substitution, and type of hydrolytically sensitive spacer present in the cross-links. This delay time was not dependent on the size of the liposomes in the range studied (0.1-0.2 microm). Microspheres which degraded more slowly showed, after a certain delay time, sustained release of the liposomes extended up to 100 days. CONCLUSIONS: A novel drug delivery concept based on the encapsulation of liposomes in biodegradable dextran-based microspheres was designed. The system released the liposomes in intact form in a controlled way after a prolonged period of time.

Calorimetry, Differential Scanning↗

PubMed2000

Subcutaneous administration of superoxide dismutase entrapped in long circulating liposomes: in vivo fate and therapeutic activity in an inflammation model.

PURPOSE: We are exploring liposomal delivery with the aim to change the pharmacokinetics and biodistribution of SOD to increase its therapeutic activity. From a practical point of view, a convenient route of administration would be the subcutaneous (s.c.) route. Liposomal size has been shown to be the most important factor influencing the rate and extent of drainage of liposomes from the s.c. injection site. METHODS: To monitor the in vivo fate of the subcutaneous administered SOD-containing liposomes in rats with a chronic arthritis inflammation, the liposomes were labeled by the co-encapsulation of the 111In-DTPA complex in the internal water space. RESULTS: Over the initial 10h-observation period post-injection, the small-sized poly(ethyleneglycol)-liposomes (mean size about 110 nm) left the site of injection to a 2-fold higher extent (45% of the injected dose) as compared to large-sized poly(ethyleneglycol)-liposomes (mean size about 450 nm). Small-sized liposomes gave a 17-fold higher uptake in the inflamed foot than the large-sized liposomes. Comparing the localization in the inflamed foot with the non-inflamed foot, uptake was more than 15-fold higher for the small-sized liposomes as compared to the large-sized liposomes. After s.c. administration, small-sized SOD-liposomes showed substantial higher activity than large-sized SOD-liposomes. S.C. administration of small-sized SOD-liposomes is equally effective as i.v. administration of the same liposomes. I.V. administration of the large-sized SOD-liposomes yielded a significantly higher activity as compared to s.c. administration. CONCLUSIONS: These results indicate that small-sized poly(ethyleneglycol)-liposomes can be used for the targeting of SOD to arthritic sites after subcutaneous administration.

Animals↗

PubMed2000

Induction of T cell anergy by liposomes with incorporated major histocompatibility complex (MHC) II/peptide complexes.

PURPOSE: The aim of this study was to use small unilamellar liposomes with incorporated MHC II/peptide complexes as a carrier system for multivalent antigen presentation to CD4 + T cells. METHODS: Purified peptide pre-loaded MHC II molecules were incorporated into small unilamellar liposomes and tested for their ability to activate A2b T cells. The outcome of T cell activation by such liposomes in the absence of accessory cells was tested via flow cytometry and a T cell anergy assay. RESULTS: Provided the presence of external co-stimulation, MHC II/ peptide liposomes were able to induce proliferation of the A2b T cell clone. More importantly incubation of these T cells with MHC II/ peptide liposomes in the absence of co-stimulation did not induce proliferation, however, a MHC/peptide ligand-density dependent down-regulation of the TCR was observed. Interestingly, when T cells after incubation with the MHC II/peptide liposomes were restimulated with their specific antigen in the presence of professional APC, these cells were anergic. CONCLUSIONS: We propose MHC II/peptide liposomes as a novel means to induce T cell anergy. The possibility to prepare 'tailor-made' liposomal formulations may provide liposomes with an important advantage for applications in immunotherapy.

Animals↗

PubMed2000

Effects of physicochemical characteristics of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes on cellular association and internalization.

The cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) is able to efficiently bind and condense DNA and to mediate transfection of a variety of cell types. In this study, fluorescence activated cell sorting (FACS), confocal laser fluorescence microscopy (CSLM) and electron microscopy (EM) techniques were used to investigate in vitro the cellular interaction of p(DMAEMA)-based polyplexes with human ovarian carcinoma cells (OVCAR-3). Cellular association and subsequent internalization only occurred when the polyplexes exhibited a positive zeta potential. Small-sized polyplexes have an advantage over large-sized complexes regarding cellular entry. The effect of the presence of tertiary amine groups versus the presence of quatenary amine groups was evaluated by comparing p(DMAEMA) with its quaternary ammonium analogue poly(2-(trimethylamino)ethyl methacrylate) (p(TMAEMA)). The combined cellular interaction and transfection results suggest that the latter polymer does not have an intrinsic endosomal escape property, in contrast to the 'proton sponge' effect proposed for p(DMAEMA). PEGylation of p(DMAEMA) effectively shielded the surface charge and yielded a notably lower degree of cellular interaction. Data on the effects of the presence of endocytosis inhibitors and an endosome-disruptive peptide in the culture medium on the cellular interaction and transfection activity of p(DMAEMA)-based polyplexes support endocytosis as being the principal pathway for intracellular delivery of plasmid. Both the CLSM and EM studies did not reveal the presence of polyplexes or plasmid outside the endocytic vesicles or within the nucleus, suggesting that intracellular trafficking from the endosomes to the nucleus is a very inefficient process.

Drug Carriers↗

PubMed1999

Liposomes in autoimmune diseases: selected applications in immunotherapy and inflammation detection.

In this contribution three examples are discussed of ongoing research where liposomes are used as carrier systems for immunotherapy and inflammation detection in autoimmune diseases. Liposomes can be used as carrier systems of antigenic peptides to peripheral blood mononuclear cells. The second example deals with their use as carrier systems for MHC-peptide complexes for multivalent Ag-presentation to autoreactive T lymphocytes to specifically modulate the activity of these T lymphocytes. The third example relates to our work on long circulating liposomes which are currently being tested in man for their potential to image inflammation sites.

Antigen-Presenting Cells↗