1、Full Terms & Conditions of access and use can be found athttps:/ Journal of Polymeric Materials andPolymeric BiomaterialsISSN: 0091-4037 (Print) 1563-535X (Online) Journal homepage: https:/ polymeric nanoparticles forefficient dual (chemo-photothermal) therapy oforal squamous carcinomaRanda H. Elshe
2、rbeny, Magda M. Hassan, Wafaa H. El-Hossary, Mona S. Shata& Wael M. DarwishTo cite this article: Randa H. Elsherbeny, Magda M. Hassan, Wafaa H. El-Hossary, Mona S.Shata & Wael M. Darwish (2020): Folate-targeted polymeric nanoparticles for efficient dual (chemo-photothermal) therapy of oral squamous
3、carcinoma, International Journal of Polymeric Materialsand Polymeric Biomaterials, DOI: 10.1080/00914037.2020.1725756To link to this article: https:/doi.org/10.1080/00914037.2020.1725756View supplementary material Published online: 17 Feb 2020.Submit your article to this journal Article views: 9View
4、 related articles View Crossmark dataFolate-targeted polymeric nanoparticles for efficient dual (chemo-photothermal)therapy of oral squamous carcinomaRanda H. Elsherbenya, Magda M. Hassana, Wafaa H. El-Hossarya, Mona S. Shataa, and Wael M. DarwishbaDepartment of Oral Pathology, Faculty of Dentistry,
5、 Suez Canal University, Ismailia, Egypt;bDepartment of Polymers and Pigments, NationalResearch Centre, Giza, Dokki, EgyptABSTRACTIn this work, the phytochemical anticancer thymoquinone (TQ) and near-infrared plasmonic goldnanorods (AuNRs) were loaded in folate-targeted pegylated poly(D,L-lactide-co-
6、glycolide) (PLGA-PEG-FA) nanocapsules. This polymeric regime exhibited in vitro sustained release kinetics of bothTQ and AuNRs. MTT assay showed the strong and synergistic chemo-photothermal effects on SCC-15 cells incubated with the combination therapy associated with dramatic ultrastructural chang
7、eson the morphology of the cells. The enhanced anticancer efficacy, as indicated by tumor regres-sion and histopathological examinations, proved the ability of the proposed regime to achievestrong synergistic anticancer effects and selective tumor targeting via dual-modal targeted system.GRAPHICAL A
8、BSTRACTARTICLE HISTORYReceived 19 October 2019Accepted 1 February 2020KEYWORDSCombination therapy; folicacid; gold nanorods; oralsquamous cellcarcinoma; PLGA1. IntroductionOral squamous cell carcinoma (OSCC) represents 90% oforal carcinoma with a quite low 5-year survival rate and isone of the 10th
9、most common causes of death in theworld1, 2. For the current treatment modalities such as sur-gery, radiotherapy, and chemotherapy, about 47% recurrencerates have been reported3. It appears at this point thatadvanced therapeutic modalities should be designed for effi-cient treatment of OSCC. Combina
10、tion therapy is a recentlyproposed strategy for efficient treatment of drug-resistanttumors. In combination therapy, two or more therapeuticsof different anticancer mechanisms are simultaneously com-posed in a biocompatible nanoprobe4. PLGA-PEG regime,recently approved by FDA, is an advantageous pla
11、tform forproduction of sustained-release delivery systems5. PLGA-PEG regime is significant in that its degradation products(lactic and glycolic acid) are easily metabolized in the bodyvia the Krebs cycle and eliminated as carbon dioxide andwater6. Furthermore, the glass transition temperature (Tg)of
12、 the PLGA copolymers is above the physiological tempera-ture of 37?C and hence they are glassy in nature, thusshows rigid chain structure and sustained release of theloaded drug in blood6. PLGA is used for production ofdrug-loaded nanocapsules of 200nm size and thus providesdrug passive targeting vi
13、a enhanced permeability and reten-tion (EPR) effect7. Active targeting can be also added tothe design via attachment of the surface of the nanoprobesto molecules of which receptors are over expressed at theCONTACT Wael M. DDepartment of Polymers and Pigments, National Research Centre, Giza, Dokki, E
14、gypt.Supplemental data for this article can be accessed on the publishers website.? 2020 Taylor & Francis Group, LLCINTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALShttps:/doi.org/10.1080/00914037.2020.1725756tumor surface8. Plasmonic photothermal therapy (PPTT), aprocess invol
15、ving administration of gold nanorods and a sub-sequent eradication with near infrared (NIR) laser, has showna high potential for treatment of many cancers9, 10. The pro-duced heat causes an ablation of the tumor cells while limitingdamage to surrounding tissues11, 12. The NIR region of thespectrum i
16、s characterized by maximal penetration throughthe body due to relatively lower scattering and absorptionfromthetissuesandbodyfluidsinthisregion13.Thymoquinone (TQ) is a natural product derived from themedicinal plant Nigella sativa and known for its high anti-inflammatory and anti-cancer activities1
17、4. However, noveldelivery strategies are still needed to enhance the biodistribu-tion and pharmacokinetics of this hydrophobic drug.In this work, we described a well-designed (chemo-pho-tothermal) combination therapeutic model designated foran efficient treatment of OSCC. This model is prepared byco
18、-encapsulation of chemotherapeutic drug TQ and plas-monic photothermal AuNRs in a folate-targeted PLGA-PEG nanocapsules. This model is designed to target keypathways in a characteristically synergistic or an additivemanner. Combination of two functional species of two dif-ferent anti-cancer mechanis
19、ms is expected to confer highanti-cancer efficacy, if a synergistic action and selectivetargeting could be achieved. Passive targeting due to EPReffect of the nanocapsules and active targeting due to fol-ate at the surface of the nanocapsules are expected toenhance the biodistribution, pharmacokinet
20、ics, and anti-tumor efficacy of the prepared nanocapsules. In vitro andvivo studies were carried out to evaluate the efficacy ofthisdual(chemo-photothermal)therapeuticmodelfortreatment of oral squamous cell carcinoma, chemicallyinduced in golden hamster, using a small, portable, inex-pensive near-in
21、frared (NIR) laser.2. Experimental2.1. MaterialsAll solvents were of analytical grade, purified and driedaccordingtointernationalstandardprocedures15.TQ,chloroauric acid (HAuCl4?4H2O, 99.99%), silver nitrate,poly(ethylene glycol) methyl ether thiol (mPEG-SH) (MW 6000),N-(3-dimethylaminopropyl)-3-eth
22、ylcarbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS), folicacid, sodium borohydride (NaBH4, 99%), 1,2-dimethylbenz-a-anthracene (DMBA) (cat. no: D3254), poly(D,L-lactide-co-glycolide)acidterminatedMw7000-17000,andL-ascorbicacidwereproductsofSigma-AldrichCo.(Steinheim,Germany).Hexadecyltrime
23、thylammoniumbromide(CTAB,98%)wasaproductofFluka.Poly(ethylene glycol) bis(3-aminopropyl) terminated, Mn?1,500(H2NPEGNH2)waspurchasedfromACROSORGANICS, Belgium. Milli-Q water was used for the prep-aration of the final formulations, otherwise deionized waterwas used. Sterilized tubing with (MWCO) of 2
24、kDa and1214kDa were purchased from Cellulose Dialysis Tubing,Fisherbrand, USA.2.2. InstrumentationDifferential scanning calorimetry (DSC) measurements werecarried out under nitrogen on SDT Q600 V20.9 Build 20with heating rate 10?C min?1. Transmission electron micro-scope (TEM) images were recorded o
25、n a JEM-2100, Jeolelectron microscope. Dynamic light scattering (DLS) instru-ment (PSS, Santa Barbara, CA, USA), using the 632nm lineof a He-Ne laser as the incident light with angel 90?andZeta potential with external angel 18.9?. Ground state elec-tronic (UV-VIS-NIR) absorption spectra were recorde
26、d atroom temperature using a computerized recording on Cary300 spectrophotometer, Agilent Technologies. Estimation ofgold was carried out using a double beam flame atomicabsorption, Agilent 240FS equipped with cross flow nebu-lizer. A vortex mixer-SA7, SA8, STAURT, US was used toenhance the encapsul
27、ation of the drug. For c counting,c-Scintillation counter (Scaler Ratemeter SR7, Nuclear enter-prises LTD, USA fitted with well type NaI(TI) crystal)was utilized.2.3. Synthesis2.3.1. Preparation of PEGylated gold nanorods (AuNRs)AuNRs were synthesized in an aqueous solution using aseed-mediated grow
28、th method16. To coat AuNRs withPEG, a final concentration of 10mM mPEG-SH and 1nMcolloidal AuNRs were mixed. AuNRs were sonicated over-night, centrifuged (13,000rpm for 15min) and redispersedin deionized water to remove non-bound PEG molecules.PEGylated AuNRs were then sterilized by filtration (0.22
29、mmpore size filter). AuNRs prepared by this method areapproximately 12nm in width and 48nm length (?4.0aspect ratio), with a longitudinal plasmon absorption max-imum at 808nm. Extinction spectra of gold suspensionsshowed no peak shift, broadening, or reduction over a one-week period prior to injecti
30、on.2.3.2. Preparation of (PLGA-PEG) and (PLGA-NH-PEG-NH-FA)PLGA-PEG conjugates were prepared using a modificationof the procedures previously described for EDC/NHS carbo-diimide chemistry17. Firstly, the carboxylic group of PLGA-COOHwasactivatedasfollows:PLGA-COOH(1g,0.0526mmol) and four molar exces
31、s of NHS (24.168mg,0.210mmol) and EDC (40.257mg, 0.210mmol) were dis-solved in methylene chloride previously dried over anhyd-rous calcium chloride. This mixture was stirred under staticdry argon for 3h at room temperature. Secondly, this solu-tion was then added dropwise to a mixture of 5 molarexce
32、ss of (NH2-PEG-NH2) and a drop of TEA in methylenechloride. After 1h stirring at room temperature, about20mL of ice-cold diethyl ether was added to give off-whiteprecipitate of (PLGA-NH-PEG-NH2). This precipitate wasdissolved in the least amount of DMSO and dialyzed againstdeionized water for 3days
33、to remove excess PEG. PLGA-PEG-FA conjugates were prepared by DCC/NHS chemistry.In brief, folic acid (39.73mg, 0.09mmol) was dissolved in2R. H. ELSHERBENY ET AL.10ml of anhydrous DMSO and then a mixture of NHS(20.72mg, 0.18mmol) and EDC (34.51mg, 0.18mmol) wasadded. The yellow mixture was stirred un
34、der static argonatmosphere for 24h at RT. Afterward, PLGA-PEG conjugate(500mg, 0.0233mmol) was added and the mixture wasstirred for 24h at RT. About 60mL of deionized water wasadded to precipitate excess unreacted FA. The later was sep-arated by centrifugation at 4000rpm for 10min as a yellowsolid.
35、The supernatant containing (PLGA-PEG-FA) conju-gate (Figure 1) was dialyzes against DI water and freeze-dried.TheproductwassubjectedtoGelPermeationChromatography (GPC) using Sephadex G-50 column andDMF as eluent.2.3.3. Formulation of TQ-loaded nanocapsules(TQPLGA-PEG-FA) nanocapsules were prepared u
36、sing amodifiedproceduretowater-oilemulsionsolvent-evaporation method18. In brief, TQ (1.2mg/mL dissolvedin 125mL of anhydrous chloroform) was mixed with either(PLGA-PEG) or (PLGA-PEG-FA) (24mg/mL dissolved in125mL of anhydrous chloroform). This mixture was stepwiseadded to a strongly stirred solutio
37、n of PVA (2%, in 2mL ofpurified water).The mixture was vortexed for 3min and theformed emulsion was sonicated (25W, 20 cycles) using amicrotip-probe sonicator while the sample was cooled overan ice bath. The organic solvent was then evaporated underreduced pressure at room temperature. Large particl
38、es couldbe easily removed by centrifugation (4000rpm, 5min.)whereas; the small and uniform nanocapsules could be sepa-ratedbycollectedusinghigh-speedcentrifugation(13,000rpm, 20min) and finally washed twice with PBS (pH7.4). To remove non-encapsulated TQ, the micelles were fil-trated through a 0.45m
39、m filter19.2.3.4. Formulation of AuNR-loaded nanocapsules(AuNRPLGA-PEG-FA) nanocapsules were prepared as fol-lows: A solution of either (PLGA-PEG) or (PLGA-PEG-FA)(24mg/ml dissolved in 250mL of anhydrous chloroform)was stepwise added to a strongly stirred AuNRs colloid(10mg/mL). After vortexing for
40、3min, the organic solventwas evaporated under reduced pressure. The nanocapsules(AuNRPLGA-PEG) or (AuNRPLGA-PEG-FA) were col-lected using high-speed centrifugation (13,500rpm, 30min)and finally washed twice with PBS (pH 7.4).2.3.5. Formulation of (TQ/AuNRs)-loaded nanocapsules(TQ/AuNRPLGA-PEG-FA) na
41、nocapsules were prepared asfollows: TQ (1.2mg/mL dissolved in 125mL of anhydrouschloroform) was mixed with either (PLGA-PEG) or (PLGA-PEG-FA) (24mg/mL dissolved in 125mL of anhydrouschloroform). This mixture was stepwise added to a stronglystirred AuNRs colloid (10mg/mL). After vortexing for 3min,th
42、e organic solvent was evaporated under reduced pressure.Thenanocapsules(TQ/AuNRPLGA-PEG)or(TQ/AuNRPLGA-PEG-FA) were collected using high-speedcentrifugation (13,500rpm, 30min) and finally washed twicewith PBS (pH 7.4).2.4. Encapsulation efficiencyThe entrapment efficiency (EE, %) of the prepared nan
43、opar-ticles was calculated according to Eq. 1. The free drug wasseparated from the nanoparticles by filtration/centrifugationtechnique using Nanosep (100kDa cut off) membrane fil-ter20. TQ concentration was assessed by the standard spec-troscopic method21,22. Thus, 1mL of ethyl alcohol wasadded to t
44、he filtrate and the absorbance at TQ prominentpeak at 257nm was assessed. A calibration curve for TQ dis-solved in the same medium was previously done (R20.99421 over the range between 1 and 25lg/mL).Encapsulation efficiency% drug?total? drug?freedrug?total? 100(1)Figure 1. Structures of TQ (top) an
45、d (PLGA-PEG-FA) conjugates (bottom).INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS AND POLYMERIC BIOMATERIALS32.5. In-vitro release kineticsThe release profile of TQ from the nanoparticles was carriedout according to the standard method reported in litera-ture23. In brief, 20mg of the lyophilized nano
46、particles wasdissolved in 10mL of PBS (pH 7.4) and placed at 37?C in ashaker incubator. At different time points (0.5, 1, 1.5, 2, and3h), the suspension was centrifuged at 10,000rpm for 5minand the supernatant was collected. Afterward, the nanopar-ticles were resuspended in fresh buffer and further
47、incu-bated. The supernatant was extracted twice with chloroformand TQ content was analyzed by the same spectroscopicmethod in the previous section. Drug release was then cal-culated according to Eq. 2.Drug Release% Conc: of drug in dialysateConc: of drug in NCs? 100(2)2.6. In vitro cytotoxicity assa
48、ysHumanoralsquamouscellcarcinoma(SCC-15)wasobtained from American Type Culture Collection (ATCC).Cells were cultured in A 1:1 mixture of Dulbeccos modifiedEagles medium (DMEM) and Hams F12 medium contain-ing 1.2g/L sodium bicarbonate, 2.5mM ofL-glutamine,15mM HEPES and 0.5mM sodium pyruvate suppleme
49、ntedwith 400ng/mL hydrocortisone, 90%; fetal bovine serum,10% at 37?C under 5% CO224, 25. After removal of themedium, cells were rinsed with 0.25% trypsin, 0.03% EDTAsolution. The solution was removed and about 2mL oftrypsinEDTA solution was added. Cells were then incu-bated at 37?C under 5% CO2unti
50、l the cells detach. Freshculture medium was added and cells were dispensed intoculture flasks. For MTT assay, cells were seeded at a densityof 104cells/well in a 96-well plate with 100lL of DMEM for24h. Different nanoformulations were then incubated to theculture medium. For laser irradiation experi
