Syntese af Gold Nanopartikel Integrated Foto-responsive Liposomer og måling af deres Mikroboble Kavitation på Pulse Laser Excitation
Summary
Denne protokol beskriver en simpel fremstillingsmetode for guld nanopartikler integreret foto-responsiv liposomer med de kommercielt tilgængelige materialer. Det viser også, hvordan man kan måle mikroboble kavitation processen med de syntetiserede liposomer ved behandling af pulseret laser.
Abstract
Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.
Introduction
Muligheden for at udløse medikamentfrigivelse brug af eksterne stimuli er en attraktiv måde at levere narkotika i spatial-, temporal- og dosering-kontrollerede mode med maksimeret specificitet og minimale bivirkninger. Blandt en lang række eksogene stimuli-responsive systemer (lys, magnetfelt, ultralyd, mikrobølge stråling), lys-udløst platforme er attraktive på grund af deres ikke-invasiv, enkelhed og tilpasningsevne i klinikkerne. 1 Omfattende forskning i det seneste årti har givet en bred vifte af platform teknologier, såsom nær-infrarødt lys ansvarlig guld (Au) nanocages belagt med smarte polymerer, 2 foto-labile, polymere nanopartikler (NPS) konjugeret med narkotika, 3 og selv-samlet porphysome nanovesicles. 4 Men disse teknologier er stadig i de præklinisk udvikling, og kræver en klar forståelse og optimering af parametre, der er involveret i processen med at iværksætte og contrullende lægemiddelstoffrigivelsen.
En af de enkleste og let tilgængelige metoder til fremstilling af et sådant system er at integrere Au nationale parlamenter med termisk følsomme liposomer 5,6, som begge er almindeligt tilgængelige på markedet og er blevet grundigt undersøgt i prækliniske og endda kliniske forsøg. Uanset begrænsningen af dybtliggende væv aktivering af Au NP'er på deres plasmoniske bølgelængde, sammenlignet med nær-infrarød-aktiveret Au nanostrukturer (f.eks nanocages), dette stadig lover godt, når de anvendes i små dyr eller til topisk levering i mennesker. 7 Der er nogle tidlige indsats i at kombinere Au nationale parlamenter med liposomer for lys-udløst frigivelse. 8-11 Mens de fleste af dem fokuserer på den nyhed af materialer, skal behandles tilgængelighed og skalerbarhed spørgsmål. Desuden rapporter om mekanismer release bruger disse nanocarriers er stadig begrænsede.
Heri betyder fremstillingen af fotofølsomtliposomer, samtidig lastet med narkotika og hydrofile Au NP'er er blevet beskrevet. Calcein anvendes som model forbindelse at evaluere indkapslingseffektivitet og frigivelsesprofilen for systemet. Desuden i dette system, lys absorberes af Au NP'er spreder til det omgivende mikromiljø i form af varme, hvilket resulterer i en stigning i den lokale temperatur. Luftmikrobobler frembringes, medens laser opvarmning og forårsage mekanisk forstyrrelse af liposomer (figur 1). Mekanismen af mikroboble kavitation bekræftes af hydrofonsystemer målinger.
Protocol
Representative Results
Discussion
Tyndfilm hydrering er den konventionelle metode til fremstilling af liposomer. Organiske opløsningsmidler (chloroform i dette tilfælde) blev først anvendt til at opløse lipiderne og derefter fjernet i en rotationsfordamper ved 37 ° C til frembringelse af et lipid tynd film på kolben. Denne lipidfilm blev hydratiseret med den vandige opløsning indeholdende 60 mM calcein og 5 nm Au NP'er. Under hydrering proces blev temperaturen opretholdt omkring 50 ° C, og kolben blev konstant omrørt ved kolben roterer. Nø…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev delvist støttet af Tier-1 Academic Research fondene ved Singapore Undervisningsministeriet (RG 64/12 til CX) og NTU-Northwestern Institute of nanomedicin.
Materials
| 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti Polar Lipids (Alabama, US) | 850355P | Powder, Store at -20 °C |
| 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) | Avanti Polar Lipids (Alabama, US) | 855675P | Powder, Store at -20 °C |
| 1, 2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000) | Avanti Polar Lipids (Alabama, US) | 880120P | Powder, Store at -20 °C |
| Gold Nanoparticles | Sigma Aldrich | 752568-100mL | 5nm particles, stabilized at 0.1mM PBS |
| Calcein | Sigma Aldrich | C0875-10g | 60mM, pH 7.4 – adjusted using NaOH |
| phosphate buffered saline (PBS) | Sigma Aldrich | P5493 | 0.1 mM, pH 7.4 |
| Double distilled water | Millipore Milli-DI water purification system | ||
| Triton X100 | Sigma, Life Sciences | X-100 | To disrupt the liposomes to calculate total encapsulation |
| Rotavapor | Buchi (Switzerland) | R 210 | Used for Lipososme preparation |
| Heating bath | Buchi (Switzerland) | B 491 | Used for Lipososme preparation |
| Vacuum Controller | Buchi (Switzerland) | V-850 | Used for Lipososme preparation |
| Vacuum Pump | Buchi (Switzerland) | V-700 | Used for Lipososme preparation |
| Recirculation bath with temperature controller | Polyscience | Used for Lipososme preparation | |
| Mini-extruder assembly with heating block | Avanti Polar Lipids (Alabama, US) | 610000 | Used for extrusion of liposomes |
| Syringes, 1000 uL | Avanti Polar Lipids (Alabama, US) | 610017 | Used for extrusion of liposomes |
| Polycarbonate filter membrane, 200nm | Whatmann | 800281 | Used for extrusion of liposomes |
| Filter Support | Avanti Polar Lipids (Alabama, US) | 610014 | Used for extrusion of liposomes |
| PD 10 Desalting coulumns, Sephadex G-25 medium | GE Healthcare, Life sciences | 17-0851-01 | Used to purify the liposomes |
| Centrifuge | Sigma Laboratory Centrifuges | 3K30 | Used to concentrate the liposomal solution |
| Rotor | Sigma | 19777-H | Used to concentrate the liposomal solution |
| Zetasizer | Nano ZS Malvern | Used for the determination of liposome size and zetapotential | |
| UV- Visible Spectrophotometer | Shimadzu | UV-2450 | Used to measure the absorbance of the samples |
| Fluorescent Spectrofluorometer | Molecular Devices | SpectraMax M5 | Used to measure the fluorescence emission of the samples |
| Nd:YAG Laser | NewWave Research | 532 nm; Maximum power: 17mJ; Width: 406 ns; Used for sample irradiation | |
| HNR Hydrophone | ONDA | HNR-1000 | 1000 mm diameter and 450 nV/Pa sensitivity, Proper working frequency range: 0.25-10 MHz; Calibration: 50 mV/Bar; Used to measure the acoustic signals |
| Digital Osciloscope | LECORY – Wave Runner 64Xi-A | Frequency: 600 MHz; Max sample rate : 10 Gs/s (at two channel); Used to record the measured acoustic signals |
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