Påvisning af Endotoxin i Nano-formuleringer bruger Limulus Amoebocyte Lysate (LAL) Assays
Summary
Påvisning af endotoksiner i industrielt fremstillet nanomateriale repræsenterer en af de store udfordringer inden for Nanomedicin. Vi præsenterer her, et casestudie, der beskriver rammerne består af tre forskellige LAL formater til at vurdere potentielle endotoxin forurening i nanopartikler.
Abstract
Når de findes i farmaceutiske produkter, en Gram-negative bakterielle cellevæg komponent endotoxin (ofte også kaldet LPS) kan forårsage betændelse, feber, hypo- eller hypertension, og i ekstreme tilfælde kan føre til væv og orgel skader, der kan blive fatal. Mængder af endotoxin i farmaceutiske produkter, derfor er strengt reguleret. Blandt de metoder, der er til rådighed for endotoxin påvisning og kvantificering, er Limulus Amoebocyte Lysate (LAL) assay almindeligt anvendt over hele verden. Mens enhver farmaceutisk produkt, der kan forstyrre LAL assay, udgør nano-formuleringer en særlig udfordring på grund af deres kompleksitet. Formålet med dette dokument er at give en praktisk vejledning for forskere uerfarne i estimeringen af endotoksiner i nanomaterialer og nanopartikel-formuleret narkotika. Heri, diskuteres praktiske anbefalinger til at udføre tre LAL formater herunder turbiditet, kromogent og gel-blodprop assays. Disse assays kan bruges til at bestemme endotoxin forurening i nanoteknologi-baserede lægemidler, vacciner og adjuvanser.
Introduction
En endotoxin er en byggesten af Gram-negative bakterielle cellevæg1,2. Det kan aktivere immunceller på meget lav (pikogram) koncentrationer1,2. Proinflammatoriske mæglere (cytokiner, leukotriener, eicosanoider, etc.) produceret af celler som reaktion på en endotoxin er ansvarlig for feber, hypotension, hypertension og mere alvorlige sundhedsmæssige problemer, herunder flere organsvigt 1 , 2 , 3. sværhedsgraden af immun-medieret bivirkninger udløst af endotoxin afhænger dens potens bestemmes af endotoxin sammensætning og struktur og måles i internationale endotoxin enheder (IUs eller EUs)3. Antallet af disse enheder pr. kg kropsvægt bruges til at indstille en tærskel pyrogen dosis af endotoxin. Denne dosis er 5 EU/kg for lægemidler administreres via alle ruter men ruten Intratekal. Lægemidler doseres pr kvadratmeter af kroppens overflade, intraokulært væsker, radioaktive lægemidler og produkter administreres via Intratekal rute har en forskellige pyrogen tærskeldosis, som er 100 EU/m2, 0,2 EU/mL, 175 EU/V (hvor V er den volumenet af det produkt, der er beregnet til administration), 0,2 EU/kg, henholdsvis4. Flere detaljer om den pyrogen tærskeldosis for forskellige lægemidler og enheder leveres og diskuteret andetsteds4,5,6.
Dyr varierer meget i deres følsomhed over for endotoxin-medierede reaktioner. Mennesker, ikke-menneskelige primater og kaniner er blandt de mest ekstremt følsomme over for endotoksiner3arter. For at undgå endotoxin-medieret bivirkninger hos patienter og forebygge urigtige konklusioner af prækliniske toksicitet og effekt studier, er det nødvendigt at præcist at detektere og kvantificere endotoksiner i både kliniske og prækliniske grade formuleringer. Flere i øjeblikket tilgængelige metoder kan opnå denne opgave. En af dem er Limulus Amoebocyte Lysate (LAL) analysen, som er almindeligt anvendt over hele verden til skærmen biomedicinske produkter for den potentielle endotoxin forurening samt at opdage bakterieinfektioner7,8,9. Den lysate er fremstillet af amoebocytes, cellerne i blod fra horseshoe krabber Limulus Polyfem bosiddende i øst-kysten af det europæiske kontinent Nordamerika7. Interessant, der er et par forskellige arter af horseshoe krabber (Tachypleus gigas og Tachypleus tridentatus) i Asien10. Tachypleus Amoebocyte Lysate (TAL) er brugt i flere asiatiske lande til påvisning af endotoxin svarende til hvordan LAL bruges i andre cuntries10. Lysates (LAL og TAL) indeholder en gruppe af proteiner, der ved aktivering giver protease aktivitet. En af disse proteiner, den såkaldte faktor C aktiveres ved kontakt med endotoxin. Aktiveret faktor C kløver Factor B, som til gengæld også bliver en protease og kløver et pro-koagulation enzym til at producere en blodpropper enzym. Resultatet af denne kæde af reaktioner er dannelsen af en gel, en stigning i stikprøven Turbiditet og i overværelse af et kromogent substrat, udseendet af en farvet produkt, der tjener som et fundament for gel-blodprop, turbiditet og kromogent assays, henholdsvis. Mens der er ingen obligatoriske LAL format, US Food and Drug Administration (FDA) forklarer i retningslinjer for baseret industrien dokument, at beslutningen om foretages i tilfælde af uoverensstemmelse i testresultater mellem forskellige LAL formater, på gel-blodprop assay5 .
Mange almindeligt anvendte laboratoriekemikalier (fx., EDTA) og kendte lægemiddel produkter (fx penicillin) forstyrrer LAL-undersøgelser,11. Indblanding identificeres normalt ved at vurdere inddrivelse af endotoxin standard aks på en kendt koncentration i en løsning, der indeholder testmateriale. Hvis spike opsving er mindre end 50% eller mere end 200%, så resultatet af LAL assay for er den givne prøvemateriale ugyldig på grund af hæmning eller ekstraudstyr, henholdsvis4. Nanoteknologi-baserede formuleringer er ofte komplekse og forstyrre LAL gennem en række mekanismer12,13,14. Mange tilgange er blevet beskrevet til at overvinde indblanding: prøve rekonstituering i specifikke buffere og overfladeaktive stoffer, protein inaktiveres ved opvarmning, ødelæggelse af lipid-baserede hule materialer ved opvarmning og supplere prøve med overskud divalent kationer5,12,13,14,15. Alternative metoder til situationer, hvor LAL indblanding ikke kan overvindes er også blevet beskrevet: ELISA, en HEK-TLR4 reporter celle line assay og massespektrometri16,17,18, 19.
Heri, er eksperimentelle procedurer for udførelse af gel-blodprop, turbiditet og kromogent LAL assays beskrevet. Disse analyser er også tilgængelige på nanoteknologi karakterisering Lab (NCL) hjemmeside20 i protokoller STE1.2 (turbiditet LAL), STE1.3 (gel-blodprop LAL) og STE1.4 (kromogent LAL). Det anbefales at foretage mindst to forskellige formater for at karakterisere den samme nano-formulering. Når resultaterne af Turbiditet og kromogent LAL er uenige, anses gel-blodprop resultater5. Når resultaterne af to LAL formater er uenige, gennemført yderligere undersøgelser, enten monocyt aktivering test (MAT) eller kanin pyrogen test (RPT) til at kontrollere LAL resultater er21. Det er vigtigt at bemærke, at hver metode bruges til registrering af endotoxin og sikkerhedsforsøg vurdering har fordele og begrænsninger21,22,23,24. Erkendelse af begrænsninger af den procedure, der anvendes til at karakterisere en given nanoteknologi formulering er afgørende for at opnå videnskabelig begrundelse for anvendelse af proceduren for optimal for at nano-formulering.
I denne undersøgelse, blev pegyleret liposomal doxorubicin brugt som en model nanopartikel formulering. Denne formulering er godkendt af det amerikanske FDA i 1995 og anvendes til behandling af kræft patienter på verdensplan25.
Protocol
Representative Results
Discussion
Oplysningerne i denne protokol er blevet beskrevet før15,26 og bygger på flere lovgivningsmæssige dokumenter udgivet af US Food and Drug Administration (US FDA eller FDA) og Amerikas Forenede Stater farmakopé (USP)4 , 5 , 6 , 27, og er også tilgængelig på NCL hjemmeside20 i protokoller STE1.2 (turbiditet L…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Undersøgelsen blev støttet af føderale midler fra National Cancer Institute, National Institutes of Health, under kontrakt HHSN261200800001E. Indholdet af denne publikation afspejler ikke nødvendigvis synspunkter eller politikker af Department of Health og Human Services, og heller ikke nævner af firmanavne, kommercielle produkter, eller organisationer indebærer godkendelse af den amerikanske regering.
Materials
| Turbidity LAL Assay | |||
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL Reagent | Associates of Cape Cod | T0051 | This reagent can be used with turbidity assay only |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 mL | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
| Chromogenic LAL Assay | |||
| Pyrochrome LAL Reagent | Associates of Cape Cod | CG1500-5 | This reagent is specific to the Chromogenic Assay |
| Control Endotoxin Standard | Associates of Cape Cod | EC010 | This standard is different than that used for turbidity and gel-clot LALs; it is optimized for optimal performance in the chromogenic assay |
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 ml | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
| Gel-Clot LAL Assay | |||
| LAL Reagent | Associates of Cape Cod | G5003 | This reagent is specific to the gel-clot assay |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 10 x 75 mm | Associates of Cape Cod | TS050 | These tubes are for use with the gel-clot assay |
| Pyrogen-free tips with volumes 0.25 and 1 mL | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Water bath, 37 C | any brand | Any brand can be used, however, it is important either to switch off water circulation or use non-circualting water bath because water flow will affect clot formation and lead to false-negative results |
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