http://informahealthcare.com/phdISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, Early Online: 1–8! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2013.867447

RESEARCH ARTICLE

Vitamin B12-loaded solid lipid nanoparticles as a drug carrier in cancertherapy

Lutfi Genc1,2, H. Mehtap Kutlu3, and Gamze Guney4

1Department of Pharmaceutical Technology, Faculty of Pharmacy, 2Plant, Drug and Scientific Researches Center (AUBIBAM), 3Department of

Biology, Faculty of Science, Anadolu University, Eskisehir, Turkey, and 4Department of Medical Biology, Faculty of Medicine, Sakarya University,

Sakarya, Turkey

Abstract

Nanostructure-mediated drug delivery, a key technology for the realization of nanomedicine,has the potential to improve drug bioavailability, ameliorate release deviation of drugmolecules and enable precision drug targeting. Due to their multifunctional properties, solidlipid nanoparticles (SLNs) have received great attention of scientists to find a solution to cancer.Vitamin supplements may contribute to a reduction in the risk of cancer. Vitamin B12 hasseveral characteristics that make it an attractive entity for cancer treatment and possibletherapeutic applications. The aim of this study was to produce B12-loaded SLNs (B12-SLNs) anddetermine the cytotoxic effects of B12-SLNs on H-Ras 5RP7 and NIH/3T3 control cell line. Resultsobtained by MTT assay, transmission electron and confocal microscopy showed that B12-loadedSLNs are more effective than free vitamin B12 on cancer cells. In addition, characterizationstudies indicate that while the average diameter of the B12 was about 650 nm, B12-SLNs wereabout 200 nm and the drug release efficiency of vit. B12 by means of SLNs increased up to 3 h.These observations point to the fact that B12-SLNs could be used as carrier systems due to thetherapeutic effects on cancer.

Keywords

Cancer, drug delivery systems, solid lipidnanoparticles, vitamin B12

History

Received 1 October 2013Revised 12 November 2013Accepted 14 November 2013Published online 17 December 2013

Introduction

Nanotechnology is a medical advantage for diagnosis, treatment,prevention and cure of cancer disease. The use of drug deliverysystems has revolutionized cancer treatment. Cancer patients usevarious anticancer drugs but these are less effective and havemajor side effects1,2. An ideal targeted drug delivery approachdoes not only increase therapeutic efficacy of drugs but alsodecreases the toxicity associated with the drugs to allow lowerdoses of the drug to be used in therapy3,4.

SLNs were developed as an alternative carrier system forpharmaceutical applications. SLNs combine the advantages andavoid the disadvantages of the other colloidal carriers (liposomes,polymeric micro- and nanoparticles)5. SLNs can increase theentry of agents into cells or tissues because of small size, toimprove the bioavailability of drugs by increasing their diffusionthrough biological membranes and to protect them againstenzyme activation which leads to a toxic product. SLNs haveunique properties such as small size, large surface area, high drugloading, a potential wide application spectrum and the avoidanceof using organic solvents during preparation6–9.

All living cells require vit. B12 (cobalamin) for survival. Insidethe cell, it is converted to two active cofactors, adenosylcobala-mine and methylcobalamine. Adenosylcobalamine is a cofactorfor methylmalonyl-CoA mutase, while methylcobalamine is acofactor for methionine synthase. B12 functions as coenzymes inthe synthesis of purines and thymidylate for DNA synthesis and isinvolved in DNA methylation. Thus, it is not surprising that

rapidly dividing tissues requiring methionine and thymidine forcell proliferation have an increased demand of cobalamin10–13.Deficiency of B12 may result in misincorporation of uracil intoDNA, leading to chromosome breaks and disruption of DNArepair that may interfere with DNA methylation and synthesis,and may cause genetic instability. This can lead to cancer14–16.

B12 displays anticancer effect against different cancer cell linesin literature. Nevertheless, the incorporation of B12 into SLNs andthe effect on 5RP7 (H-ras transformed-rat embryonic fibroblast)and NIH/3T3 (mouse embryonic fibroblast) cell culture (controlgroup) have not been reported. The purpose of the present study isto evaluate the efficacy of B12-loaded SLNs compared with freeB12 in the treatment of cancer. Physico-chemical properties offree B12 and B12-SLNs, such as size and polydispersity index (PI)have been measured by photon correlation spectroscopy (PCS)and also zeta potential has been measured by Zetasizer Nano ZS(Malvern Instrument, Malvern, UK). Evaluation of drug release(buffer solution at pH 7.4 and 6.8) and entrapment efficacy wascarried out. Next, the cytotoxic effects were examined on H-Ras5RP7 and NIH/3T3 cells. The structure and ultrastructure changesof these cells were determined by using transmission electronmicroscopy (TEM) and confocal microscopy. The results clearlydemonstrate that B12-SLNs are significantly more effective in thetreatment of cancer as compared with free B12.

Materials and methods

Materials

Compritol� and polyoxyethylene sorbitan monooleate (Tween 80)were purchased from Merck Schuchardt (Darmstadt, Germany)and Acros Organics (Morris Plains, NJ). Vitamin B12, Dulbecco’s

Address for correspondence: Gamze Guney, Department of MedicalBiology, Faculty of Medicine, Sakarya University, Sakarya, Turkey.E-mail: gamzeguney@anadolu.edu.tr

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Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS),penicillin, streptomycin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe-nyl-2 H-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO)and acridine orange were obtained from Sigma Aldrich (St. Louis,MO). All other chemicals used were analytical grade. Allsolutions were formulated using double distilled water.

Cell culture

A control cell line NIH/3T3 (mouse embryonic fibroblasts) and5RP7 (H-ras transformed rat embryonic fibroblast) cancer cellline were used in these experiments. They were obtained fromThe American Type Culture Collection (ATCC). Frozen stockvials of the cells were thawed and used. Cells were routinelycultured at 37 �C in a humidified atmosphere with 5% CO2 (inair), in culture flasks containing DMEM, supplemented with 10%FBS, L-glutamine (1 mM final concentration), and penicillin/streptomycin at 100 units/mL.

Production of SLNs

The hot homogenization technique was chosen for preparation ofSLNs described by Muller and Lucks17. SLN formulations consistof 5% Compritol� as a lipid, 2.5% Tween 80 as a surface activeagent and 2% vitamin B12 as an active ingredient. In order toprepare SLNs, the lipid phase was melted at 10� 1 �C above themelting point of the solid lipid and the temperature was set to80 �C in a thermostated water bath during the stirring18. Aftermelting of the lipids, vitamin B12 was added to lipids at the sametemperature. To this mixtures, Tween 80 was added slowlythrough Ultra-Turrax (T25, Janke&Kunkel IKA�, Germany) at20 500 rpm and cooled down to room temperature. After thisprocesses, SLNs were stored at 4 �C in small glass vials withoutlight exposure over a period of 3 months19.

Particle size and zeta potential measurement

The average diameter and PI of B12 and B12-SLNs weredetermined by PCS using a Zetasizer Nano ZS (MalvernInstrument, Malvern, UK) at 25 �C, respectively. Before meas-urement, each sample had to be diluted with distilled water to anadequate intensity (n¼ 3). To determine particle size, zetapotential and PI, the concentration of B12 was 25 mg/mL.

Zeta potential values of the SLNs and vitamin B12 weredetermined by using Zetasizer Nano ZS (Malvern Instrument,Malvern, UK). Analyses were performed in disposable capillarygreen zeta cells at 25 �C by diluting the samples. Conductivity ofdistilled water was adjusted to 50mS with NaCl to avoid anyconductivity changes (n¼ 3).

HPLC assay

The analytical process validation, method Q2(R1) of theInternational Harmonization Committee was used and theparameters such as linearity, accuracy, precision and specificitywere evaluated20.

B12 and B12-SLNs were analyzed by Agilent 1100 seriesHPLC (Agilent, Santa Clara, CA) on a C18 column (25� 2.5 cm,5mm). The mobile phase was a mixture of methanol–water(30:70, v:v) with a flow rate of 0.8 mL/min. UV detection for vit.B12 was conducted at 361 nm. The retention time was about7.3 min. HPLC method was validated.

Drug release and entrapment efficiency (%EE) capacity

Drug release was determined by measuring the absorbance ofeach sample at 361 nm. 2.5 mg/mL of B12-SLNs and B12 weredispersed in 400 mL of phosphate-buffered saline (PBS) at pH 6.8and 400 mL distilled water at pH 7.4 and kept at 37 �C� 1 �C on a

water bath under mechanical stirring (100 rpm). At suitable timeintervals, samples were analyzed using HPLC method (n¼ 3). Inthe meantime, an equal volume of the blank medium at the sametemperature was added to keep volume constant.

In order to evaluate the amount of vit. B12 entrapped intonanoparticles, 1.25 mg/mL vit. B12-SLNs were dissolved in 10 mLof methanol. Then the solution was centrifuged at 5000 rpm for15 min at 4 �C. Then the samples dispersed in PBS (1:5, v:v) werecentrifuged at 5000 rpm for 15 min. Finally, the sample wasanalyzed by HPLC. Before injecting to HPLC, both B12 and B12-SLN formulations firstly solved and then filtered to avoiddamaging to column and this process is conventional application.

MTT assay

The cytotoxicity of vit. B12 and B12-SLNs against H-Ras 5RP7 andNIH/3T3 cells was assessed by using MTT assay. The MTT assay isbased on the protocol described for the first time by Mossmann(1983)21. The cells were seeded at 1� 105 cell/mL per well into 96well plates following incubation for adherence at 37 �C in 5% CO2.Then the cells were incubated in free B12 or B12-SLNs for a series ofdifferent drug concentrations at 24, 48 and 72 h. Then 20 mL MTTsolution (5 mg/mL) was added to each well and the plates wereincubated for 3 h. The medium in each well was changed by 200mLDMSO and mixed thoroughly to dissolve the dark blue crystals for10 min at room temperature in order to ensure all crystals could bedissolved. The plates were read with ELISA reader (EL X 808),using test wavelength of 540 nm (n¼ 3).

Transmission electron microscopy

The uptake of B12-SLNs and B12 by NIH/3T3 and H-Ras 5RP7cancer cells and nanoparticle size and morphology weredetermined using a TEM (TEM FEI Tecnai BioTWIN,Hillsboro, OR). TEM is a method of probing the microstructureof SLNs. Initially, in order to examine morphology of B12-SLNs,they were dispersed directly into the distilled water. Then theseformulations were poured out Cu grid coated several times. Afterbeing stained by 2% osmium tetroxide and dried under roomtemperature, the sample was ready for TEM investigation. H-Ras5RP7 and NIH/3T3 cells grown in DMEM medium were fixedwith 2.5% glutaraldehyde in 0.1 M PBS at pH 7.4 and left in PBSovernight atþ 4 �C. After being embedded in agar and postfixation in 2% osmium tetroxide, cells were dehydrated in gradedethanol: 70%, 90%, 96% and 100%. Then the cells were embeddedin EPON 812 epoxy and sectioned on ultramicrotome (LEICAEM UC6, Wetzlar, Germany). They were thin sectioned using aglass knife to a maximum thickness of 100 nm. The sections werestained with lead citrate and uranyl acetate.

Confocal microscopy

H-Ras 5RP7 and NIH/3T3 cells with the most effective concen-tration of the B12 and B12-SLNs (20mM) were incubated for 24 hat 37 �C. After incubation, growth medium was removed and cellswere washed with PBS and were fixed with 2% gluteraldehyde for15 min at room temperature. Then the cells were washed withPBS and stained with acridine orange.

Morphological changes of cells were showed by a Leica TCS-SP5 II confocal microscope and the supplied software (LeicaConfocal Software Version 2.00, Wetzlar, Germany). The laserpower did not exceed usually 10% of the maximum value.

Results

Characterization of SLNs

Particle size and zeta potential with PI of B12 and B12-SLNs weremeasured. The average diameter of B12 was about 650 nm and the

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PI was average 0.5. The average diameter of B12-SLNs was about200 nm, the PI was 0.3 and the zeta potential was� 5 mV as givenin Table 1. Additionally, the size of this nanoparticle approved byusing TEM was about 210 nm (Figure 1).

After three months, we measured particle size, PI and zetapotential of B12-SLNs. We did not show any differences comparedwith previous measurement. Thus, these formulations were foundto be stable.

HPLC assay

Five milligrams of B12 were accurately weighted and dissolved inmethanol and volume was adjusted to 5 mL. Five samples of25mL, 50 mL, 250 mL, 500mL and 1000mL were taken from thisstock solution and diluted to 5 mL with methanol. In order toestablish linearity, a minimum of five different concentrations arerecommended. The stock solutions of B12 in mobile phase wereused to prepare five sample solutions of varying B12

concentration.Validated HPLC method was performed for the determination

of B12. The linearity, precision and repeatability values of themethod were calculated. Excellent linearity was obtained for B12

between concentration of 5 and 200mg/mL (5, 10, 50, 100,200mg/mL) in mobile phase and R2¼ 0.9998. The repeatability ofthe method was checked by the analysis of six replicate injectionsof B12 in mobile phase using three different concentrations: 5(low), 50 (medium) and 200 (high) mg/mL to provide suitableinterval. Measurements performed on three different concentra-tions (low, medium, high) for evaluating the repeatability andreproducibility of the analytical method have been used to verifythe precision of the method. Because the coefficient of variation% is below 2%, the method precision was found to be withinthe targeted intervals for repeatability and reproducibility test.

These analyses were repeated throughout 3 d. The repeatability ofthe method was expressed as the relative standard deviation(RSD; coefficient of variation) of measured concentration. RSDvalues were less than 1%. The limit of detection–limit ofquantification (LOD–LOQ) values of HPLC method werecalculated. LOQ values of B12 were found to be 4.92 mg/mLwhile LOD values were 1.64 mg/mL (n¼ 6). The lowest concen-tration level used in this method is 5 mg/mL. Because LOD andLOQ values are less than this concentration, it can be concludedthat our method is sensitive.

Drug release and %EE capacity

In order to evaluate the possibility of B12 release of loaded B12-SLNs, drug release profile was investigated in PBS at pH 7.4 andpH 6.8 (Figure 2). The release of free B12 was lower from thenanoparticles dispersion. Incorporation of nanoparticle dispersiondecreased the drug release. The rate at which vit. B12 was releasedfrom SLNs was affected by the pH of dissolution medium; therelease rate increased as the pH decreased. As shown in Figure 2,after 30 min, the amount of B12 released was 80% in pH 6.8,whereas B12-SLN was released nearly 100% within 3 h. Besides,B12 was released 80% from SLN formulation in pH 7.4 within 3 h.These results indicate that SLN extended the drug release timerelated to vit. B12. Thus, SLN formulations are found suitable forsustained release of vit. B12.

EE% values of B12-SLNs are also reported. The EE% ofnanoparticles were evaluated by dissolving methanol, then dilutedPBS and subsequent HPLC analysis of the solution. A highamount of drug could be incorporated in the SLNs. Drug assayshowed 91–93% content of total amount. Ninety-two percent ofB12 was incorporated in the SLNs. The result showed that B12 hasa high entrapment efficacy in SLNs.

(A) (B)

Figure 1. TEM images of B12-loaded SLNs. (A)� 43 000 and (B)� 87 000.

Table 1. Particle size, polydispersity index and zeta potential of B12-loaded SLN prepared by hot homogenization.

Particlesize (nm) PI

Zeta potential(�mV)

Particle size(nm) (90 d) PI (90 d)

Zeta potential(�mV) (90 d)

199.3� 5 0.398� 0.08 �5.13� 2 196.3� 4.2 0.404� 0.10 �5.55� 1.8198.8� 7 0.385� 0.06 �5.60� 1.7 199.5� 3.4 0.375� 0.08 �5.90� 1.9200.7� 8 0.402� 0.06 �5.28� 2.1 205.8� 6.5 0.385� 0.02 �5.35� 2.1

DOI: 10.3109/10837450.2013.867447 Vitamin B12-loaded SLNs 3

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Cytotoxicity of B12 and B12-SLNs

The in-vitro results from MTT assay showed that the inhibition offree B12 was weaker than the B12-SLNs group (Figure 3A, B) on5RP7 cells. The results showed that survival of 5RP7 cells

decreased in a dose-dependent manner in the presence of eitherfree B12 or B12-SLNs (2.5–20 mM) in Figure 3(A, B). Both freeB12 and B12-SLNs incubated with NIH/3T3 cells for 24–72 h didnot show any effect on the viability of NIH/3T3 cells even at thehighest concentration in Figure 3(C, D). But the inhibitory effect

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Figure 3. Effects of B12-loaded SLNs and vit. B12 (2.5–20mM) on viability of 5RP7 after 24, 48 and 72 h exposure in (A) and (B). Cytotoxic effects ofB12 and B12-loaded SLNs on 3T3 cells after 24, 48 and 72 h exposure in (C) and (D).

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of B12-SLNs was significantly stronger, with maximal inhibitionat 20 mM of 53% in H-Ras 5RP7 cells.

Images of laser scanning confocal microscopy

Effects of the B12 and B12-SLNs on the morphology of H-Ras5RP7 and NIH/3T3 cells were shown in Figure 4. H-Ras 5RP7and NIH/3T3 cells were stained with acridine orange andobserved under excitation at 488 nm. In Figure 4, H-Ras 5RP7cells treated with B12-SLNs were smaller and round in shapeand their cytoplasm especially nuclei were stained moredensely than NIH/3T3 cells. The effects of B12-SLNs weredamaged connection between cells. The nuclei of H-Ras 5RP7control cells were flat while that of B12-SLNs treated cellswere coarse and B12-SLNs entered the cells. In Figure 4(B),H-Ras 5RP7 cells treated with B12 were larger and moreflattened in shape than H-Ras 5RP7 control cells. InFigure 4(E, F), NIH/3T3 cells showed no morphologicalchanges. In the NIH/3T3 control group cells, the nucleus wasbigger and smoother than that of NIH/3T3 cells treated by vit.B12 and B12-SLNs.

Images of transmission electron microscopy

Effects of the B12 and B12-SLNs on the structure and ultrastruc-ture changes of H-Ras 5RP7 and NIH/3T3 cells were investigated(Figure 5). Control cells showed their normal shape and surfacemorphology under TEM Figure 5(A). As seen in Figure 5(C),exposure of H-ras transformed cells to B12-SLNs showedshrinkage and destroying mitochondrial membrane and increasedvacuoles. B12-SLNs entered the tumor cells. In Figure 5(E, F), aspredicted from the cytotoxicity experiments, TEM observations ofNIH/3T3 cells showed that cellular structures were well preservedwith no visible abnormalities, following incubation with both B12

and B12-SLNs but we observed increased number of mitochondriaand deformation mitochondrial membrane.

Discussion

During cancer treatments, many patients use dietary supplements,particularly antioxidants, in the hope of reducing the toxicity ofchemotherapy and radiotherapy22. Low systemic levels of B12 andfolate are found to be associated with the increase in cancerbecause deficiency of B12 and folate is known to influence

(B)(A) (C)

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Figure 4. Confocal images of 5RP7 cells after 24-h incubation. (A) Nontreated cells, (B) treated with vit. B12 and (C) treated with B12-loaded SLNs.Confocal images of NIH/3T3 cells after 24 h incubation. (D) Nontreated cells in their normal fibroblast shape, (E) treated with vit. B12 and (F) treatedwith B12-loaded SLNs.

DOI: 10.3109/10837450.2013.867447 Vitamin B12-loaded SLNs 5

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mutation rate in cells by virtue of their role either as antioxidant,cofactor in DNA metabolism or as important intermediates insynthesis of nucleotides required for DNA replication andrepair10,14,15. Therefore, plasma B12 and folate levels might

have an important role in the process of neck, head, leukemia,pancreatic, colorectal and cervical cancer23–28.

Colloidal nanoparticle systems incorporating anticancer agentscan overcome resistance to drug circulation, increase accumula-tion of drug in cancer cells and reduce toxicity towards normalcells29,30. Previous studies have shown that solid lipid nanotech-nology is available in literature which described preparationtechniques (hot and cold homogenization, ultrasonication, micro-emulsion and solvent evaporation methods) characterization (byusing Zeta Sizer Nano ZS, NMR, HPLC, LC-MS, FT-IR), thestructural features of research, formulation and storage, drugloading properties and drug release characteristics31–34. Inprevious studies, various anti-carcinogenic substances (mitoxan-trone, tamoxifen, cholesterylbutyrate, butyrate, doxorubicin andpodophyllotoxin) loaded SLNs were studied in various cancercells (HT-29, MCF-7, HL-60, etc.). As a result of these studies,chemotherapeutic drugs-loaded SLNs display significant cytotox-icity or have an effective long-term anti-cancer effect againstcancer cell lines35–40.

Vit. B12 is necessary for cell proliferation but excessive andunnecessary use of vitamins can cause some adverse effects. Toovercome these problems and improve the pharmacologicalprofile, we formulated drug-loaded SLNs, using a vit. B12 and

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Figure 5. TEM images of 5RP7 and NIH/3T3 cells after 24-h incubation. (A) Nontreated 5RP7 cells (�16 500). (B) Images of 5RP7 cells which isgiven vit. B12 (�8200). (C) Images of 5RP7 cells which is given vit. B12-loaded SLNs (�8200). (N: nucleus, V: vacuole).! Apoptotic blebbing, B12-SLNs. (D) Nontreated NIH/3T3 cells (�6000). (E) Images of NIH/3T3 cells which is given vit. B12 (�6000). (F) Images of NIH/3T3 cells which isgiven B12-loaded SLN (�8200). (N: nucleus; ! vacuoles and mitochondria).

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Figure 6. TEM images of B12-loaded SLNs in 5RP7 cells after 24 hincubation. (A) �43 000. (B) �16 500. ! Lamellar body.

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lipid matrix. The system improves B12 efficacy and much morereduces the viability H-Ras 5RP7 cell line as shown inFigure 3(A, B).

Physical–chemical properties (size, zeta potential, PI) are quitecritical for biopharmaceutical behavior of SLNs. As shown inTable 1, B12-SLNs that produced hot homogenization techniquegained a relative good stability and dispersion quality41. Besides,drug release of B12-SLNs was slower than free vit. B12 (Figure 2).The release rate decreased for SLNs with a higher lipidconcentration, which has been explained by the physical morph-ology of the lipid particles41,42. Therefore, it was revealed thatSLNs produced by this method could achieve high drugincorporation.

In addition, the specificity of uptake, internalization andlocation of B12 and B12-SLNs were studied and compared in twodifferent cell lines, H-Ras 5RP7 and NIH/3T3 (control cell line).The B12-SLNs were dispersedly localized in the cytoplasm andcytoplasmic nucleus membrane while free B12 was diffuselylocalized outside the cells as depicted in Figure 4(B, C). As shownFigure 5(C, F), the nanoparticles entered the cell by passivediffusion and accumulated in the cytoplasm. Phagocytic vacuolesand lamellar body were formed and cells underwent apoptosis ashighlighted in Figure 6. The enhanced effects of B12-SLNs maybe related to the rapid internalization in the cytoplasmicdepartment, followed by the drug release of SLNs inside thecells, thus enhancing anticancer effects on H-Ras 5RP7 cells.

Conclusion

SLNs have a great potential to cure cancer, with the least sideeffects. B12-SLNs could be prepared successfully promising theiruse for cancer treatment. The result of this study reveals that B12-SLNs may be used as a carrier system for cancer therapy. Thisstudy may be improved by using different formulation ofnanoparticles, different preparation techniques and differentcancer cells. Future drug uptake studies in in-vivo model willdemonstrate the potential efficacy of SLNs to deliver cancer. So,the use of nanoparticles as drug delivery vehicles for anticancertherapeutics has great potential to revolutionize the future ofcancer therapy.

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