RESUMO
In photodynamic therapy (PDT), photosensitizers and light are used to cause photochemically induced cell death. The selectivity and the effectiveness of the phototoxicity in cancer can be increased by a specific uptake of the photosensitizer into tumor cells. A promising target for this goal is the folic acid receptor α (FRα), which is overexpressed on the surface of many tumor cells and mediates an endocytotic uptake. Here, we describe a polysaccharide-based nanoparticle system suitable for targeted uptake and its photochemical and photobiological characterization. The photosensitizer 5, 10, 15, 20-tetraphenyl-21H, 23H-porphyrine (TPP) was encapsulated in spermine- and acetal-modified dextran (SpAcDex) nanoparticles and conjugated with folic acid (FA) on the surface [SpAcDex(TPP)-FA]. The particles are successfully taken up by human HeLa-KB cells, and a light-induced cytotoxicity is observable. An excess of free folate as the competitor for the FRα-mediated uptake inhibits the phototoxicity. In conclusion, folate-modified SpAcDex particles are a promising drug delivery system for a tumor cell targeted photodynamic therapy.
RESUMO
Therapeutic vaccination is and remains a major challenge, particularly in cancer treatment. In this process, the effective activation of dendritic cells by a combination of distinctly acting adjuvants and an antigen is crucial for success. While most common vaccine formulations lack the efficiency to trigger sufficient T cell responses in a therapeutic tumor treatment, nanovaccines offer unique properties to tackle that challenge. Here, we report the stepwise development of a nanocapsule for vaccination approaches, comprising a shell consisting of antigen and loaded with a superadditive adjuvant combination. In a first initial step, we identified the combination of resiquimod (R848) and muramyl dipeptide (MDP) to have a superadditive stimulatory potential. Particulated in Spermine-modified dextran-nanoparticles, the dual-adjuvant maintains its superadditive character and stimulates murine dendritic cells (DC) stronger than the soluble equivalents. The second step was to evaluate a protein-based nanocapsule as suitable antigen source for the induction of antigen-specific T cell responses. Therefore, the DC-mediated antigen-specific T cell proliferation upon treatment with nanocapsules, whose shell consists of ovalbumin (OVA), was assessed. At least, the superadditive adjuvant combination was encapsulated into OVA-nanocapsules to create the final nanovaccine. Its immunostimulatory potential for DC was extensively tested by measuring the expression of co-stimulatory surface markers, the secretion of pro-inflammatory cytokines and the capability to mediate OVA-specific T cell responses. The developed nanovaccine triggers strong superadditive dendritic cell stimulation and potent antigen-specific CD4+ and CD8+ T cell proliferation. Combined with a high modifiability, an excellent biocompatibility, low cytotoxicity and an enormous loading capacity, the introduced antigen-nanocapsule provides an enormous potential for the effective delivery of superadditive adjuvant combinations, particularly when they target intracellular receptors.
Assuntos
Adjuvantes Farmacêuticos/química , Antígenos/química , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Nanocápsulas/química , Ovalbumina/química , Linfócitos T/imunologia , Acetilmuramil-Alanil-Isoglutamina/química , Adjuvantes Farmacêuticos/administração & dosagem , Animais , Antígenos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Citocinas/metabolismo , Dextranos/química , Humanos , Imidazóis/química , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Espermina/químicaRESUMO
Surface modifications of nanoparticles can alter their physical and biological properties significantly. They effect particle aggregation, circulation times, and cellular uptake. This is particularly critical for the interaction with primary immune cells due to their important role in particle processing. We can show that the introduction of a hydrophilic PEG layer on the surface of the polysaccharide-based nanoparticles prevents unwanted aggregation under physiological conditions and decreases unspecific cell uptake in different primary immune cell types. The opposite effect can be observed with a parallel-performed introduction of a layer of low molecular weight dextran (3.5 and 5 kDa) on the particle surface (DEXylation) that encourages the nanoparticle uptake by antigen-presenting cells like macrophages and dendritic cells. Binding of DEXylated particles to these immune cells results in an upregulation of surface maturation markers and elevated production of proinflammatory cytokines, reflecting cell activation. Hence, DEXylated particles can potentially be used for passive targeting of antigen presenting cells with inherent adjuvant function for future immunotherapeutic applications.
Assuntos
Células Dendríticas/efeitos dos fármacos , Dextranos/farmacologia , Macrófagos/efeitos dos fármacos , Nanopartículas/química , Polietilenoglicóis/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Dextranos/química , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polietilenoglicóis/química , Cultura Primária de Células , Baço/citologia , Propriedades de Superfície , Regulação para CimaRESUMO
AIM: Evaluation of dextran-based nanoparticles (DNP) as a drug delivery system to target myeloid cells of the liver. MATERIALS & METHODS: DNP were synthesized and optionally PEGylated. Their toxicity and cellular uptake were studied in vitro. Empty and siRNA-carrying DNP were tested in vivo with regard to biodistribution and cellular uptake. RESULTS: In vitro, DNP were taken up by cells of the myeloid lineage without compromising their viability. In vivo, empty and siRNA-carrying DNP distributed to the liver where a single treatment addressed approximately 70% of macrophages and dendritic cells. Serum parameters indicated no in vivo toxicity. CONCLUSION: DNP are multifunctional liver-specific drug carriers which lack toxic side effects and may be utilized in clinical applications targeting liver macrophages.
Assuntos
Dextranos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Células 3T3 , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Sobrevivência Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/toxicidade , Tamanho da Partícula , Polietilenoglicóis/química , Células RAW 264.7 , Propriedades de Superfície , Distribuição TecidualRESUMO
We propose a structure-based protocol for the development of customized covalent inhibitors. Starting from a known inhibitor, in the first and second steps appropriate substituents of the warhead are selected on the basis of quantum mechanical (QM) computations and hybrid approaches combining QM with molecular mechanics (QM/MM). In the third step the recognition unit is optimized using docking approaches for the noncovalent complex. These predictions are finally verified by QM/MM or molecular dynamic simulations. The applicability of our approach is successfully demonstrated by the design of reversible covalent vinylsulfone-based inhibitors for rhodesain. The examples show that our approach is sufficiently accurate to identify compounds with the desired properties but also to exclude nonpromising ones.
RESUMO
Buruli ulcer is a neglected disease caused by Mycobacterium ulcerans and represents the world's third most common mycobacterial infection. It produces the polyketide toxins, mycolactones A, B, C and D, which induce apoptosis and necrosis. Clinical symptoms are subcutaneous nodules, papules, plaques and ulcerating oedemae, which can enlarge and destroy nerves and blood vessels and even invade bones by lymphatic or haematogenous spread (osteomyelitis). Patients usually do not suffer from pain or systematic inflammation. Surgery is the treatment of choice, although recurrence is common and wide surgical excisions including healthy tissues result in significant morbidity. Antibiotic therapy with rifamycins, aminoglycosides, macrolides and quinolones also improves cure rates. Still less exploited treatment options are phytochemicals from medicinal plants used in affected countries. Vaccination against Buruli ulcer is still in its infancy.