Anisotropic Gold Nanoparticle Decorated Magnetopolymersome: An

Article pubs.acs.org/journal/abseba

Anisotropic Gold Nanoparticle Decorated Magnetopolymersome: An Advanced Nanocarrier for Targeted Photothermal Therapy and DualMode Responsive T1 MRI Imaging Ekta Roy,† Santanu Patra,† Rashmi Madhuri,*,† and Prashant K. Sharma‡ †

Department of Applied Chemistry and ‡Functional Nanomaterials Research Laboratory, Department of Applied Physics, Indian Institute of Technology (Indian School of Mines), Dhanbad, Jharkhand 826 004, India S Supporting Information *

ABSTRACT: Herein, we report the advanced polymer vesicle [made up of triblock polymer: poly(ethylene oxide)-co-poly(Cys-AuNP@FA)-copoly(3-methoxypropylacrylamide] having encapsulated magnetic nanoparticle capable of targeted methotrexate delivery (having folic acid as tagging agent), photothermal therapy [anisotropic gold nanoparticle (AuNPs)] and stimuli-responsive T1-imaging (as MRI contrast agent). The prepared polymersome, called as magnetopolymersome (MPS), after encapsulation of magnetic nanoparticle (Gd-doped) is not only high yield and simple in synthesis but also possess very high biocompatibility, more than 95% drug encapsulation efficiency and effective near-infrared (NIR) responsive photothermal therapy. The MPS is highly stable under normal physiological environments and other extreme end conditions (like presence of serum or Triton-X 100) and have excellent stimuli-responsive (temperature and NIR) T1-contrast effect in vitro conditions (60.57 mM−1 s−1). To explore the role of shape of AuNPs on the photothermal therapy and drug delivery behavior of prepared nanocarrier, herein, we have synthesized four different shapes of AuNPs, i.e., spherical, triangle, rod, and flower. It was found that nanoflowerconjugated MPS shows the most efficient NIR responsive behavior in comparison to their other colleagues, which broke the ancient myth that spherical nanoparticle are the best candidate for drug delivery process. These features make nanoflower or other anisotropic nanoparticle-based polymersome a very promising and efficient nanocarrier for drug loading, delivery, imaging, and photothermal therapy. KEYWORDS: magnetopolymersome, anisotropic gold nanoparticle, photothermal therapy, targeted drug delivery, stimuli-responsive T1-contrast agent

1. INTRODUCTION From the very ancient ages, interdisciplinary nanotechnology/ biotechnology research has been devoted toward “biomimicking”, in which the structural analogues of natural products were designed and hope to behave them natural.1 For example, the polymer-based vesicles mimicking the cell membrane were called as polymersome and are very popular in the field of biotechnology and drug delivery because of their biocompatibility, biodegradability, and ability to encapsulate therapeutic molecules.2 Having polymers as backbone and/or core materials, their thicker membrane system made them more stable and robust, and therefore secured their place in various fields of biomedical applications including drug delivery,3 synthetic biology,4 and biosensing.5 The polymer membrane allowed us to design and experiment the new possibilities in this field, which may results in polymersome having higher mechanical strength, stimuli-responsive behavior, and possibility for functionalization with targeting moieties etc.6 Although, they are interesting and popular candidate for drug delivery system, their low-stability (disassembly in the © XXXX American Chemical Society

physiological conditions like high/low pH), limited chemical functionalization, matrix or dilution effects, and leak tightness have limited their efficacy.7 However, other than stability factor, another important aspect of the drug delivery system is membrane permeability, which makes the path clear and drug reached to the site. To enhance the therapeutic efficiency and avoid any toxic effects of drugs in the body, it is very important that drug should be delivered at targeted sites or prime locations, where it is actually needed. The features can be added in the polymersome by choosing the stimuli-responsive polymer segments in the vesicle. The external stimuli like temperature, pH, light, ultrasound radiation etc. can alter as well as control the self-assembly and phase transition behavior of the responsive polymersomes and leads to the triggered or controlled drug delivery. In the literature, these properties have been tried well by some of the researchers and therefore the Received: February 6, 2017 Accepted: March 21, 2017

A

DOI: 10.1021/acsbiomaterials.7b00089 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Scheme 1. (A) Chemical Reactions Showing Synthesis of Triblock Polymer; (B) Graphical Representation Exhibiting the Synthesis of Anisotropic Gold Nanoparticle Modified Magnetopolymersome

stimuli-responsive polymersomes, which respond with change in external stimuli like temperature,8 pH,9 light,10 ultrasound radiation,11 are successfully designed and employed as drug delivery system. However, this will not solve the problem, because targeting of these moieties is still lacking in the stimuliresponsive liposomes. Therefore, introduction or attachment of targeting ligands is required at the surface of polymersome. In

this respect, attachment of many ligands or targeting agents like folate,12 cell-penetrating peptides (CPPs),13 antibodies,14 sugars15 have been explored and studied in the literature. Like, Alibolandi et al. have reported folate and quantum dots conjugated polyethylene glycol conjugated poly(D,L-lacticcoglycolic acid) polymerosme for chemotherapy of cancer.12 Dongen et al. have reported enzyme filled polymersome based B


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rod, and flower) MPS. We have started the study with spherical NPs and it was found that flower-shaped AuNP-modified MPS are taken up and transported across the cells to a greater extent with efficient drug delivery compared to their other-shaped colleagues. In addition, inclusion of targeting ligand on the polymer further enhanced the cellular uptake of MPS. The morphology of the immobilized MPS was studied by transmission electron microscopy (TEM) and the hydrodynamic diameter was measured by dynamic light scattering (DLS) analysis. The proposed MPS showed a small hydrodynamic diameter (HD), good photothermal response, rapid but targeted MTX delivery, appropriate renal clearance, enhanced and dual-responsive (temperature and NIR) contrast effect for MRI and very efficient combination (chemotherapy and hyperthermic) treatment for cancer cells.

on polystyrene 40-block-poly[l-isocyanoalanine(2-thiophen-3ylethyl)amide and tagged with TAT, a cell penetrating peptide.13 Gracia et al. have reported a tumor homing peptide, iRGD functionalized pH sensitive poly(oligoethylene glycol methacrylate)-poly(2- (diisopropylamino)ethyl methacrylate) polymersome for targeted delivery of paclitaxel in tumor cells.16 To improve the existing properties of polymersome and curtail their drawbacks, recently, nanomaterials came in limelight. Among the different type and classification, the most important role have been played by magnetic and near-infrared (NIR)-responsive nanomaterials. The combination of polymersome with NIR-responsive nanomaterials such as gold nanoparticles (spherical, rod, shell and hexapods), carbon nanotubes, graphene oxide, metal nanofilms, silver nanocluster and up-conversion nanoparticles could make the deep penetration of drug carrier without exhibiting toxicity against human body.17 For example, Wang et al. have reported fluorescent porous carbon nanocapsules where fluorescent carbon dots was functionalized on the surface of porous carbon shell for a potential application in cell imaging, responsive drug delivery and NIR photothermal therapy simultaneously.18 Yu at al. have reported thermo and NIR responsive gold nanorod mediated liposome for smart-responsive drug release at tumor site.19 Similarly, incorporation of magnetic nanoparticle (MNPs) as core in the polymersome could able to introduce magnetic field targeted delivery, tumor, or cancer cell imaging via magnetic resonance imaging (MRI) or magnetic hyperthermic treatment of tumors cells.20 Like, Qin et al. have reported iron oxide incorporated self-assemble polymersome for T2-weighted MRI and drug delivery.21 It is true that introducing new moieties of a different type always precedes innovation of new formats, but it is difficult to compile all of the above properties in one single system. Therefore, it is needed to develop a novel polymersome that has a fast endocytosis rate, accelerated endosomal escape ability, high drug loading capacity, enhanced stability, and responsive targeted drug delivery. Taking this point into consideration, we have tried to design a noncytotoxic, stable, temperature-sensitive, and NIRresponsive polymersome, well equipped with nanoparticles, responsive polymer segments, and targeting moiety. The prepared polymersome possesses a core of gadolinium-doped superparamagnetic iron oxide nanoparticle (Gd-SPIONs), and is therefore termed as magnetopolymersome (MPS), which adds the dual behavior of cancer cell imaging (by MRI) and treatment (by hyperthermia) to the vesicle. In addition, the folic acid and anisotropic gold nanoparticle (AuNP)-modified temperature and NIR-responsive triblock asymmetrical copolymer [poly(ethylene oxide)-co-poly(Cys-AuNP@FA)-co-poly(3methoxy propylacrylamide) (PEO-co-PCF-co-PMA)] made the MPS a responsive, targeted, and stable nanocarrier for methotrexate (MTX). Biocompatible PEO chain is designed as the outer corona because it is stealthy to immune system. The temperature-responsive PMA is designed as the inner corona of the polymer vesicle for rapid endosomal escape. The cysteine-monomer as a central chain gives the possibility for attachment of anisotropic AuNPs and folic acid moieties at the MPS surface. According to the literature, in the design of efficient therapeutic carriers for targeted delivery, the morphology of NPs is the most important factor and may decide the fate of nanocarrier. To clarify the role of NP morphology on the performance of MPS, for the first time, we have designed the anisotropic AuNP-modified (sphere, triangle,

2. EXPERIMENTAL SECTION The synthesis strategy used of preparation of magnetopolymersome (MPS) conjugated with different shaped AuNPs is shown in Scheme 1A, B. To prepare the MPS, the first requirement is preparation of triblock polymer. In the synthesis of triblock polymer, poly(ethylene oxide) was converted into a macroinitiator, which further gets attached with other monomers in step-by-step reactions. As shown in the scheme, first the PEO-Br initiated the polymerization using Cys-Au@ FA monomer and made the block polymer, i.e., PEO-co-PCF-Br. Here, vinyl derivative of cysteine was used for conjugation of folic acid and AuNPs to the surface of MPS. The diblock polymer was further conjugated with another thermoresponsive monomer (3-methoxypropylacrylamide) results in triblock polymer. The triblock polymer is finally converted to polymer vesicles using film rehydration method. To the vesicle SPIONs were encapsulated as core resulting in magnetopolymersome (MPS). On the basis of the shape of AuNPs conjugated with cysteine monomer, the MPS is categories in four different class, i.e., gold nanosphere- (AuNSs-MPS), nanorod(AuNRs-MPS), nanotriangle- (AuNTs-MPS), and nanoflower-modified (AuNFs-MPS) MPS. Polymer vesicle without AuNPs or SPIONs is named as polymersome (synthesis process is described in Scheme S1). The synthesis and characterization of some components used in preparation of polymer, i.e., carboxymethyl cellulose-methotrexate (CMC-MTX) conjugate (Section S1), Gd-doped SPIONs (Section S2), different -haped AuNPs (Section S3), folic acid and AuNPconjugated vinyl derivative of cysteine (Cys-Au@FA, Section S4, Figures S1 and S2) are discussed in the Supporting Information. 2.1. Synthesis of PEO-Br Macroinitiator. The preparation of PEO-Br macroinitiator was accomplished by earlier reported method with slight modification.22 Herein, 2.5 mM PEO−OH was dried in a vacuum oven at 60 °C for 30 min, followed by addition of 15.0 mL tetrahydrofuran (THF). To the mixture, nitrogen gas (99.999%) was passed to create inert atmosphere and 6.25 mM of 2-bromoisobutyric acid bromide (dissolved in 1 mL dry THF) was added. After that, the reaction mixture was cooled with ice and 3.75 mM triethylamine was added. The whole mixture was placed for 48 h at room temperature and the resulting product was precipitated in hexane. The resulting compound i.e. PEO-Br macroinitiator was recrystallized in ethanol and a white crystal was obtained. The product was characterized by FT-IR and 1H NMR studies and discussed in Section S5 and Figures S3 and S4. 2.2. Synthesis of Poly(ethylene oxide)-co-poly(Cys-AuNP@ FA) Diblock Copolymer (PEO-co-PCF-Br) by Atom Transfer Radical Polymerization (ATRP) Method. To prepare poly(PEO)co-poly(Cys-Au@FA), we used a simple ATRP process. First, 2.0 g of synthesized PEO-Br macroinitiator (0.45 mM) was dissolved in anisole (3.0 mL) by stirring. After complete dissolution of initiator, 2.5 g of Cys-Au@FA and 0.45 mM bipyridyl were added to the mixture. The reaction mixture was deoxygenated with nitrogen for 30 min, followed by addition of 0.45 mM CuBr. The reaction was continued under a nitrogen atmosphere at 65 °C for 15 h, after that the dry THF was added and stirred for 5 min. THF was removed using a rotary C


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Figure 1. TEM images of (A) AuNSs, (B) AuNTs, (C) AuNRs, and (D) AuNFs. (E) UV−visible spectra of AuNSs, AuNSs@MPS, AuNTs, and AuNTs@MPS. (F) UV−visible spectra of AuNRs, AuNRs@MPS, AuNFs, and AuNFs@MPS. evaporator and CH2Cl2 was added to dissolve the block polymer, which is reprecipitated in hexane. The resulting block copolymer, i.e., PEO-co-PCF-Br was dried under vacuum at 30 °C and stored in desiccator. The product was characterized by FT-IR and 1H NMR studies and discussed in Section S6 and Figures S5 and S6. 2.3. Synthesis of Triblock Copolymer [PEO-co-PCF-co-poly(3-methoxypropylacrylamide), (PEO-co-PCF-co-PMA). To prepare PEO-co-PCF-co-PMA, we employed a similar ATRP method, in which 2.0 g of prepared block polymer (PEO-co-PCF-Br) was dissolved in 3.0 mL of anisole, followed by addition of 5.72 mM 3methoxypropylacrylamide and 0.45 mM bipyridyl. The reaction

mixture was deoxygenated, followed by addition of 0.45 mM CuBr and reacted at 65 °C for 15 h. To the resultant product was added dry THF, which was evaporated and then reprecipitated in CH2Cl2, dried under vacuum at 30 °C, and stored in a desiccator. After this, the resultant polymer containing −Br as end group was removed by hydrolysis of the product, resulting in formation of triblock polymer (PEO-co-PCF-co-PMA). For this, 2.0 g of PEO-co-PCF-co-PMA-Br was dissolved in 10 mL of CH2Cl2 and 5.0 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred at room temperature for 12 h and the excess solvent and TFA were removed by rotary evaporation. A light yellow color solid polymer was obtained, which D


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Figure 2. (A) XRD pattern of SPIONs, Gd-doped SPIONs, and AuNFs@MPS. TEM images of (B) polymersome and (C) AuNFs@MPS (inset HR-TEM: AuNFs@MPS). (D) Temperature and (E) NIR-responsive behavior of polymersome, AuNSs@MPS, AuNRs@MPS, AuNTs@MPS, and AuNFs@MPS. (F) Stability studies of polymersome, AuNSs@MPS, AuNRs@MPS, AuNTs@MPS, and AuNFs@MPS against various concentrations of Triton X-100 solution. reduced pressure to give a thin polymer film on the wall of the flask. Then 10.0 mL of either Milli-Q water or PBS was added and the resulting dispersion was stirred at room temperature (RT) overnight before being sonicated for 20 min, results in formation of polymer vesicle. After that, 1.0 mg mL−1 polymer vesicle was mixed with 0.5 mg of Gd-doped SPIONs and the reaction mixture was equilibrated at 60 °C for 3 h with stirring. After this, the nonencapsulated polymer vesicle was separated by centrifugation (3 min, 8000 rpm) and the supernatant containing MPS was used for further studies. In addition, a

was dried under vacuum for overnight, purified by dialysis against deionized water for 2 days and thereafter results in pure triblock polymer. The product was characterized by FT-IR and 1H NMR studies and discussed in Section S7 and Figures S7 and S8. 2.4. Preparation of Magnetopolymersome (MPS). To prepare magnetopolymersome (MPS), we first prepared the polymer vesicle of PEO-co-PCF-co-PMA polymer by film rehydration (Scheme 1A, B).23 Briefly, 50 mg of triblock copolymer was dissolved in 15 mL of CHCl3 in a round-bottom flask. The solvent was then evaporated under E


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ACS Biomaterials Science & Engineering control polymersome (without SPIONs or AuNPs) was also synthesized using the similar procedure but the polymer does not have AuNPs and/or SPIONs conjugation (Scheme S1). Similarly, to explore the targeting effect of folic acid, we also prepared an MPS without folic acid. For this, the PEO-co-poly(cys-AuNPs)-co-(PMA) triblock polymer was used and termed as MPS-without FA. 2.5. Drug Loading and Encapsulation Profile of MPS. In this work, methotrexate (MTX), a popular anticancer drug, was chosen as the model drug. Here, the different shaped AuNPs (AuNSs-, AuNTs-, AuNRs- and AuNFs-) modified MPS and polymersome were used to study the role of nanoparticle morphology on the drug-uptake and release profile. First, to study the drug loading capacity, the drug was mixed with the MPS/polymersome keeping drug/carrier ratio of 0.05 (w/w) and mixture was sonicated at 37 °C for 15 min. The drugloaded MPS/polymersome was removed from solution by centrifugation at 10000 rpm for 10 min and supernatant was added into the electrochemical cell (10.0 mL) for determination using square wave voltammetry technique. The encapsulation and loading efficiency was calculated by eqs 1 and 2, respectively

encapsulation efficiency = Dc / D0100

(1)

drug loading content = Dc / Dt100

(2)

similar diameters but various morphologies as the model systems to investigate the effects of shape on the performance of MNPs-containing polymersome. AuNSs were synthesized using CTAB and sodium citrate; while AuNTs were fabricate using NaBH4 as reducing agent. AuNRs were synthesized via a seed-growth method, while the morphology of nanoflower was controlled by addition of ascorbic acid. As shown in the TEM images of prepared AuNPs, the clear morphology with homogeneous distribution was obtained in all the four different shaped nanoparticles (Figure 1A−D). It can be seen that welldefined all the nanospheres, triangle, nanorods, and flowers are formed, with more than 98% nanoparticles of respective shapes in the corresponding samples. The diameter of AuNSs was found 10 nm, while AuNRs was of ∼100 nm in length and ∼10 nm in diameter. The AuNTs have the angle size of 10 nm and diameter of nanoflower was obtained as 10 nm. It is well-known that optical property of AuNPs and their characteristic peak in UV-spectrum mainly arises from surface plasmon resonance (SPR), which totally depends on the size, shape, and dielectric environment of AuNPs. Herein, also the four different shapes of AuNPs exhibit distinct SPR peaks in their UV-spectra and shown in the Figure 1E, F. Starting with spherical-shaped AuNPs, in the UV spectrum a single peak around 580 nm was observed, however, when the shapes of AuNPs get changed from spheres to rods, prism and flower, the SPR band get split into two bands. The strong band in NIR region, appear at 750, 755, and 760 nm for AuNRs, AuNTs, and AuNFs, respectively, corresponds to electron oscillations along the long axis, referred to longitudinal band. Another weak band present in the UV-region at similar wavelength to that of AuNSs, referred to the transverse bands. The presence of different peaks in UV spectrum with change in the morphology of AuNPs supports the successful synthesis of different-shaped AuNPs. 3.2. Characterization of MPS. 3.2.1. XRD. The XRD pattern of SPIONs, Gd-doped SPIONs, and AuNFs@MPS is shown in the Figure 2A. The XRD patterns of SPIONs and Gddoped SPIONs show seven characteristic peaks, at 29.94, 35.50, 43.02, 53.45, 57.05, 62.83, and 71.70°, which corresponds to the (220), (311), (400), (422), (333), (440), and (533) planes, respectively. These diffraction peaks belong to the characteristic crystalline spinal structure (JCPDS file no. 79−0418) of cubic iron oxide (Fe3O4). It was also observed that the intensity of diffraction peaks decreases markedly in the Gd-SPIONs, signifying that the crystalline character of the SPIONs had reduced because of the doping of Gd. However, after encapsulation of Gd-SPIONs to the polymer vesicle, the AuNFs@MPS shows one extra peak around 24.70°,24−26 which could be attributed to the successfully encapsulation of GdSPIONs in the polymer vesicle. 3.2.2. UV−Visible Study. Figure 1E, F shows the UV−vis spectra of different-shaped AuNP-modified MPS. As shown in the previous UV spectra of AuNPs, the AuNSs exhibited only one peak around 580 nm, which lies in the UV region of the spectrum. However, after conjugation with MPS (i.e., AuNSs@ MPS), the peak get shifted toward right side (bathochromic shift), become slightly broader and appeared at 600 nm, i.e., in the NIR region. A similar trend was found with other AuNPs, where the peak in the NIR region get shifted more toward longer wavelength and appeared at 800, 790, and 800 nm for AuNRs@MPS, AuNTs@MPS, and AuNFs@MPS, respectively. Among them, the AuNFs@MPS shows strong absorption band in the NIR region, therefore expected to exhibit better NIR

Where Dc = concentration of drug in the MPS/polymersome, D0 = total amount of drug, and Dt = total amount MPS/polymersome. The concentration of drug was determined by standard calibration equation calculated in the concentration range 0.5 to 11.0 mg L−1. The detail of electrochemical measurement is discussed in Section S8 and Figure S9. 2.6. Dual-Stimuli Triggered MTX Release. The drug release study of the drug-loaded MPS/polymersome was performed in two different conditions: (1) with variation in the temperatures, i.e., at 25 and 42 °C and (2) in the presence or absence of NIR radiation using laser source of 910 nm wavelength and power capacity of 2.0 W (Power Technology, AR, USA). 2.6.1. Temperature-Responsive MTX Release. The drug-loaded MPS/polymersome (500.0 μL) was placed in a 1.0 mL vial and the temperature of solution was increased. When the solution attained the temperature of 25 and 42 °C, the concentrations of MTX released from MPS/polymersome was measured using the electrochemical method. 2.6.2. NIR-Triggered MTX Release. For the study, 500.0 μL drugloaded MPS/polymersome was taken in a 1.0 mL vial and irradiated with the NIR laser light (placed at the distance of 5.0 cm from vial) for different periods of time intervals (up to 30 min). The solution temperatures were detected with an infrared digital thermometer (Equinox; EQ-DT530A) and concentrations of MTX released were detected by an electrochemical method. 2.7. In Vitro MRI Experiment and Magnetic Relaxivity Measurement. For the In vitro MRI experiment 3.0 T clinical MRI instrument (In the local pathology laboratory) was used. The relaxation test was performed in two different conditions: (1) variable temperature (37 and 42 °C) and (2) in the presence and absence of NIR. For the in vitro responsive relaxation test, the Gd concentration in AuNF@MPS was determined by electrochemical method. The AuNF@MPS was first taken in water and after mild shaking the solution was transferred into tubes with different concentrations for the MRI study. The conditions were varied and T1-MRI images were acquired every 5 min. The relaxation time [longitudinal (T1) and transverse (T2)] were measured for samples containing various concentrations of AuNF@MPS. The 1/T1 and 1/T2 values were plotted as a function of Gd concentration and from the slope of the curve; the longitudinal (r1) and transverse relaxivity (r2) were obtained. The experimental process for cell culture, cell viability study through MTT assay and blood clearance of AuNF@MPS has been discussed in Section S9 and S10.

3. RESULT AND DISCUSSION 3.1. Characterization of Different-Shaped AuNPs. In this work, we have first fabricated the well-defined AuNPs of F


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ACS Biomaterials Science & Engineering Table 1. Characteristics of Polymersome and AuNP-Modified Magnetopolymersomes (MPS) material polymersome AuNSs@MPS AuNTs@MPS AuNRs@MPS AuNFs@MPS a

hydrodynamic diameter at 25 °C (nm) 85.5 102.5 102.5 102.0 101.0

± ± ± ± ±

1.2 1.3 1.0 1.3 1.4

a

PDI 0.11 0.10 0.13 0.12 0.11

± ± ± ± ±

0.2 0.1 0.2 0.1 0.2

EE (%)

b

± ± ± ± ±

8.5 18.8 16.0 14.3 12.8

70.0 90.0 89.8 88.3 87.0

EC (%)

1.1c 1.1 1.1 1.1 1.1

± ± ± ± ±

0.4c 0.4 0.3 0.3 0.3

diameter at 42 °C (nm) 65.0 82.0 82.0 81.5 81.5

± ± ± ± ±

1.1 1.3 1.2 1.4 1.5

change in diameter after NIR treatment (nm) 85.0 80.0 81.5 81.0 81.0

± ± ± ± ±

1.3 1.4 1.3 1.2 1.4

EE = encapsulation efficiency. bEC = encapsulation content. cCalculated after 10 h of incubation.

Furthermore, to explore the effect of integrated AuNPs over the morphology of prepared MPS, the TEM images of AuNSs@MPS, AuNRs@MPS and AuNTs@MPS has also been recorded (Figure S12). In each image, core−shell morphology of prepared MPS is clearly visible which suggest the successful encapsulation of SPIONs in the polymer vesicles. However, the effect of AuNSs, AuNTs, and AuNFs integration with MPS is not very prominent, which showed spherical shape, but integration of AuNRs results in a slight distortion from the spherical shape of MPS. 3.2.5. Temperature-Sensitive Behavior of MPS. The temperature-sensitive/-responsive behavior of the polymersome and MPS (modified with different-shaped AuNPs) was characterized by DLS study in the temperature range of 25−45 °C. Initially, with an increase in temperature, a slight change in the diameter was observed. However, as shown in the Figure 2D, at LCST (at 42 °C) a sharp change in the hydrodynamic diameter was observed for all the prepared MPS. Above LCST value, the diameter decreases to the lowest value, owing to the conversion of hydrophilic polymer matrix into hydrophobic. It is reported that below LCST the polymer chains are swollen by uptake of water and above LCST a phase transition takes place and water is expelled and phase transition was observed.26 It was also observed that different-shaped AuNPs do not have any effect on the temperature-responsive behavior of MPS. 3.2.6. NIR Responsive Behavior of MPS. It was expected that incorporation of AuNPs to the polymersome moiety can add the NIR-responsive feature in the prepared MPS. NIR-resonant nanomaterials such as AuNPs are attractive therapeutic agents because they can deliver drugs via noninvasive hyperthermia.28 NIR light in the range of 700−900 nm has been popularly used to locally rise the cell temperature and results in the photothermal effect.29 For this, 30.0 μg L−1 of different-shaped AuNP-modified (AuNSs, AuNTs, AuNRs, and AuNFs) MPS was irradiated with an NIR-laser source (800 nm) and the change in temperature was measured. Here, water was used as a control sample. As shown in the Figure 2E, AuNFs@MPS shows a very rapid increase in the temperature (∼40 °C) in a very small time span of 3 min. However, the other-shaped AuNP-modified MPS, i.e., AuNSs, AuNTs, AuNRs, does not respond so well and shows 25.8, 27.5, and 32 °C temperature in 3 min, respectively. However, the control polymersome (prepared without AuNPs) did not show any significant rise in temperature. The study suggest that prepared MPS having AuNFs as the NIR-resonating unit could be used as NIRresponsive drug carrier, which exhibited a sufficient temperature rise in a small time duration. 3.3. Stability Study of Polymersome and MPS. In the field of drug delivery, the long-term stability of drug encapsulated polymersome is a major challenge for the researchers. Therefore, first, the stability of prepared MPS was investigated during their storage at room temperature. No

response than others. In addition to the UV spectra, the phase transition temperature of polymersome and different-shaped AuNP-modified MPS was also performed employing cloud point measurement method and found to be ∼42 °C (Section S11 and Figure S10). 3.2.3. Hydrodynamic Diameter. The hydrodynamic diameter of the prepared polymersome was also measured by DLS method and the results are portrayed in Table 1. From the table, it was observed that the size of the polymersome is around 86 nm, when SPIONs are not encapsulated to the core. After encapsulation of SPIONs, i.e., MPS, the size obviously increases and reached to around ∼100 nm, which does not changed further based on the conjugation of different shapes of AuNPs. As per reported literature, different kind of tumor vessels has different and larger pore size in comparison to the normal vessels and therefore permeability of drug carrier depends upon their size also.19 For example, maximum pore size for breast tumor vessels might be around ∼50−60 nm, whereas breast tissue has pore size of ∼5 nm only.27 In this study, the size of MPS is around 100 nm. Because of the good flexibility of the polymersome, it easily penetrated into tumor vessels (∼50−60 nm) and therefore benefited from tumor accumulation. 3.2.4. Morphological Analysis. The morphology of prepared polymersome and AuNFs@MPS was studied by transmission electron microscopy (TEM). As shown in the image (Figure 2B, C), the polymersome has a regular and spherical morphology in which the outer ring looks slightly darker than the inner circular part, which suggests the formation of a vesiclelike structure after assembly of the polymer chains. The diameter of prepared polymersome was found to be ∼70 nm. Interestingly, in the TEM image of AuNFs@MPS, the core− shell type of arrangement can be easily visualized, where the core does not have any boundary. The inner darker and outer lighter morphology of prepared AuNFs@MPS clearly supports the encapsulation of SPIONs in the polymer vesicle. The diameter of AuNFs@MPS was found to be ∼90 nm, which is slightly lower than the value reported via DLS, which exhibited the hydrodynamic diameter. To explore the nanoparticle integration with AuNFs@MPS, the high-resolution TEM (HR-TEM) images of AuNFs@MPS was also recorded and shown as Inset in Figure 2C. In the core−shell type AuNFs@MPS, the integration of small nanoparticle is clearly visual in the shell or outer layer. It can be seen from the HR-TEM image that the SPIONs core is a single crystal and is enclosed by multiple grains of the polymer shell, confirming the heterogeneous nucleation. In the shell the small nanoparticles are also visible which supports the successful integration of AuNPs with polymer shell. However, contrary to these, in the high magnification TEM image of polymersome does not showed any nanoparticle integration or core−shell type structure (Figure S11). G


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Figure 3. (A) Stability studies of polymersome, AuNSs@MPS, AuNRs@MPS, AuNTs@MPS, and AuNFs@MPS in the serum solution. (B) Plot of % encapsulation efficiency and loading capacity of polymersome, AuNSs@MPS, AuNRs@MPS, AuNTs@MPS, and AuNFs@MPS. (C) Magnetic hysteresis loop of Gd-SPIONs and AuNFs@MPS (inset: AuNFs@MPS solution in the presence of external magnet). (D) Hyperthermia plot of AuNFs@MPS and Gd-SPIONs (Inset: T1-weighted MRI images of the AuNFs@MPS in aqueous solution at various concentrations). (E) In vitro the hyperthermia effect and normalized graph of AuNFs@MPS on human fibroblast and MCF-7 cancer cells. (F) Blood clearance of the AuNFs@ MPS.

obvious change in their physical appearance and diameter occurs after 3 months of storage; however, some aggregation of particles was appeared after 3 months (Table 1). During the storage period, the encapsulation efficiency (%) was also evaluated and found that %EE of MPS remains somewhat same (∼98%) after three months interval (Table 1), whereas the

encapsulation efficiencies of control polymersome, decreases to 89−86%. The results of the stability experiments show that encapsulation of SPIONs makes the MPS more stable than the control polymersome. In a different study, the stability of MPS in the presence of Triton X-100 and serum medium was also investigated. The H


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3B, the drug encapsulation in all the MPS were increases initially with increase in the time and shows ∼95% encapsulation in 2h; however the value is only ∼70% with polymersome, after 10 h of incubation with drug. In addition, modification with different-shaped AuNPs does not cause any significant change in drug loading profile. However, it can be easily concluded that MPS have higher %EE and %EC values as compared to the control polymersome. 3.5. Magnetic Properties of MPS. 3.5.1. VSM Study. For separation and hyperthermia applications, it is important that SPIONs must retain their magnetic properties after modification with nonmagnetic polymer layer. Herein, the magnetic properties of Gd-SPIONs and AuNFs@MPS were studied by VSM study. Their room temperature magnetization curves were shown in Figure 3C. The saturation magnetization value of Gd-SPIONs and AuNFs@MPS was found to be 292.4 and 206.1 emu g−1, respectively. The magnetization value of GdSPIONs gets decreased, after modification with polymer matrix, which is obvious due to the nonmagnetic nature of polymers. However, the resulting slightly lower magnetization value of AuNFs@MPS is sufficient enough for hyperthermia treatment. The coercivity in hysteresis loops is negligible in all the samples, implying these materials were superparamagnetic.31 3.5.2. Magnetic Capture Test. The camera image of welldispersed AuNFs@MPS (1.0 mg mL−1 in water), with external magnet is shown in Figure 3C (inset). As depicted, upon application of an external magnet to the vial, the AuNFs@MPS were collected near the magnet within 10 s of time span and the solution become colorless, clear, and transparent. To simulate the path and role of AuNFs@MPS inside the blood vessel, in a different set of experiments, we transferred the AuNFs@MPS from one end of a thin plastic tube to the other with the help of a laboratory used normal magnet (Scheme S2). It was found that when AuNFs@MPS was injected at one end of the tube (assumed as blood vessel); it travels to another end by the magnetic field generated via the magnet fixed at the other end of tube. The AuNFs@MPS get collected at one place of tube (near to the magnet) and the clear solution (without AuNFs@ MPS) was collected in the vial attached at another end of tube. Almost 99% of the nanoparticle gets accumulated around the magnet in 1 min by traveling the distance of 35 cm. The experiment suggests the targeting of AuNFs@MPS at the required site with fast speed and high specificity. 3.5.3. In Vitro Hyperthermia Treatment. To study the magnetic heat-generation characteristics of Gd-SPIONs and AuNFs@MPS, we dispersed the nanoparticles in water and subjected them to alternating magnetic field (AMF). As shown in Figure 3D, with increasing magnetic treatment a sharp increase in the temperature was observed with respect to time. The initial temperature of the suspension was 20 °C, which rose to 75 and 70 °C for Gd-SPIONs and AuNFs@MPS, respectively, after 2 min of magnetic treatment. After modification with nonmagnetic polymer, slightly fewer rises in the temperature was observed in case of AuNFs@MPS. However, the temperature rise in the prepared AuNFs@MPS is sufficient enough for effective hyperthermia treatment of cancer cells, i.e., 45 °C with in a time span of 40 s.32 To investigate the selective heating efficiency of AuNFs@ MPS under AMF, we conducted an in vitro hyperthermia experiment against the MCF-7 cells and normal human fibroblasts. Figure 3E shows that the AuNFs@MPS was nontoxic to normal human fibroblasts and MCF-7 cells in the absence of magnetic field and similar to the control

multilayer stability study was done against Triton X-100, which is a nonionic surfactant, has a hydrophilic poly(ethylene oxide) chain and an aromatic hydrocarbon hydrophobic group. It is reported that when Triton X-100 interacts with polymersome/ MPS, the vesicle−micelle transition involved and induces the solubilization of lipid vesicles causing deformation of membrane like structure.30 DLS study was used to measure the change in diameter of MPS in the presence of different concentrations of Triton X-100. As shown in Figure 2F, the average diameter of the control polymersome gradually decreased by Triton X-100, indicating deformation of the membrane. However, the MPS show a minimum change in their diameter, despite the addition of Triton X-100. This may be due to the encapsulation of MNPs in the core of vesicle, which increases the physical stability. To further analyze the stability of prepared MPS, their serum stability was also studied and shown in the Figure 3A. For this, the MPS were incubated in the DMEM and their hydrodynamic diameters were measured by DLS. As shown in the image, slight change in the diameter of control polymersome was observed with change in the time. However, an almost negligible change in the diameter was obtained for MPS. The stability study successfully supports the higher stability of the newly synthesized polymersome in comparison to the control one. In some of the literature, to verify the stability of prepared polymersome/MPS solution, their critical aggregation concentration (CAC) was also studied and calculated.30 CAC is the concentration above which the intermolecular hydrogen bonding, micelles, or other aggregates start forming, i.e., the lower the CAC, the stronger the micelles upon dilution. For such a purpose, pyrene was used as a probe molecule. This CAC point could be calculated by finding the point of intersection of the tangents of fluorescence curve shown in Figure S13 and the CAC values of AuNFs-, AuNRs-, AuNTs-, AuNSs@MPS, and control polymersome were determined to be about 56.23, 56.00, 53.7, 51.00, and 52.00 mg L−1, respectively. The values suggest their stability in the aqueous medium also. 3.4. Drug Loading Study. The drug loading capacity of polymersome/MPS was studied by thin film/sonication method. In Table 1, the poly dispersity index (PDI), % encapsulation efficiency (EE), and %encapsulation content (EC) of all prepared MPS (AuNSs-, AuNRs-, AuNTs-, and AuNFs@MPS) and control polymersome were displayed. All the prepared MPS/polymersomes have shown good homogeneity, with PDIs less than 0.20 and %EE and %EC values in the range of 70−90% and 8.5−18.2%. The values of %EE and %EC of MPS were calculated after 2 h of incubation of drug; however, for the polymersome, the value was calculated for 10 h incubation. Prior to study, the variation in drug loading capacity of different-shaped AuNP-modified MPS with time and effect of MTX concentration variation on the loading capacity of AuNFs@MPS was also studied (Figure S14). Herein, different concentrations of MTX in the range of 0.5− 11.0 mg L−1 were incubated with AuNFs@MPS for 2h and corresponding %EE was calculated. As shown in the figure, 9.0 mg L−1 concertation of MTX is sufficient to achieve the 95% encapsulation efficiency. Therefore, 9.0 mg L−1 concentration of MTX has been optimized to calculate the %EE and %EC of different-shaped AuNP-modified MPS and polymersome. The %EE and %EC of different shaped AuNP-modified MPS and polymersome has been calculated to explore the drug loading capacity of prepared carriers. As shown in the Figure I


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Figure 4. Drug release profile of MTX-loaded polymersome and MPS: (A) at 25 °C and (B) 37 and 42 °C temperatures. (C) Drug release profile of MTX-loaded polymersome and MPS in the presence and absence of NIR radiation. (D) Drug release profile of MTX-loaded polymersome and AuNFs@MPS with different power density source of NIR. (E) Cell viability study of polymersome and different-shaped AuNP-modified MPS. (F) Cell viability study of MTX and MTX-loaded MPS in the absence of NIR radiation.

polymersome. However, under the AMF the AuNFs@MPS is almost nontoxic to the normal cell and shows a considerable amount of cytotoxicity against MCF-7 cells. From the studies, it can be concluded that under AMF, the AuNFs@MPS selective heat generated in MCF-7 cell was sufficient to kill the cancer cells. Toxicity of AuNFs@MPS toward cancer cells and nontoxicity toward normal cells could be explained on the basis of

interaction between cells and drug carrier. According to the literature, the high-affinity folate receptor (FR) has more restricted expression pattern in normal tissues but is often highly expressed in tumor tissues including breast, ovarian, cervical, brain, nasopharyngeal, colon, renal, and lung.33 This increased expression of FR likely supports the increased need for folate by the rapidly dividing tumor cells. The FR is not accessible to the bloodstream in normal tissues because of its J


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ACS Biomaterials Science & Engineering localization to the apical surface of polarized epithelium.33 However, transformed or cancer cells lose their polarization, thus making FR available to agents introduced via the bloodstream. Therefore, with high-level expression in many tumors, FR has become a highly attractive molecule for directed tumor targeting. Therefore, many groups, including ours, have used folic acid to functionalize the prepared MPS or polymersome to selectively target tumor cells. 3.5.4. Stimuli-Responsive T1-MRI Performance. To evaluate the effectiveness of the AuNFs@MPS as T1-MRI contrast agent, we performed the magnetic resonance relaxivity study on a 3.0 T human clinical scanner by immersing the different concentrations of AuNFs@MPS at different temperatures (37 and 42 °C) in presence or absence of NIR radiation. The concentration-dependent longitudinal relaxation rate (1/T1) and transverse relaxation rate (1/T2) of AuNFs@MPS was summarized in Figure 3F. The calculated longitudinal and transversal relaxivity values (r1 and r2, respectively) for AuNFs@MPS were tabulated in the Table S1. The longitudinal relaxivity r1 and transverse relaxivity r2 of prepared AuNFs@ MPS were calculated from the linear fitting of 1/T1 or 1/T2 plot versus concertation of Gd (mM) present in the AuNFs@ MPS (Figure 3F) and estimated to be 60.57 and 200.0 mM−1 s−1, respectively. The AuNFs@MPS synthesized in this work clearly exhibit rather high r1 value with relatively low r2/r1 ratios, i.e., 3.30. The low value of r2/r1 indicates that the prepared AuNFs@MPS can be used as T1 MRI contrast agents.26 However, it was also observed that MRI signal enhancement took place with addition of stimulus like temperature or NIR. The r1 value get significantly enhanced and increased to 96.28 mM s−1 (at 42 °C) and 101.14 mM s−1 (after incubation with NIR for 2 min). Although the exact explanation for all these enhancements requires further researches, however, according to the literature, there are two major factors influencing T1 relaxivity value (r1) of Gd based T1 contrast agents. The first factor is the number of surface Gd3+ ions, which is commonly perceived as the surface-to-volume ratio (S/V). Small NPs possess larger S/V and in general the r1 values increased with decreasing particle size. The second factor is the distance between the paramagnetic center and the surrounding water proton. Therefore, it can be concluded that the substantially enhanced relaxivity should be mainly due to the direct exposure of a large amount and evenly distributed Gd ions to the water molecule in the polymersome.34 As a result, the high sensitivities to temperature and NIR bestow the AuNFs@MPS with a great potential to act as both temperatureresponsive and NIR-responsive T1-MRI contrast agent. Figure 3D (Inset) shows the MR assays of a T1-weighted imaging phantom which present excellent positive T1 contrast enhancement. As depicted in the image, the MR signals in T1weighted contrast change brightened as the concentration of Gd ions increased, showing a clear dose-dependent color change, owing to the increase in relaxation of water protons with the increase in concentration dose.35 Furthermore, to explore the role of AuNFs@MPS in MRI, their relaxivity was also compared with commercially available MRI contrast agent and portrayed in Table S1. As shown in the Table, the AuNFs@MPS could able to produce effective T1 contrast enhancement effects, which were several times higher than the r1 values of various commercial T1 contrast agent reported in the literatures.36 Thus, it can be concluded that the prepared MPS@AuNFs was determined to be suitable for MRI applications as effective T1 contrast agents. The as-synthesized

MPS@AuNFs has multiple functionalities applicable for multimodal imaging of cancer cells. To support this, the in vitro imaging of MPS@AuNFs was demonstrated in the MCF7 cells (Figure S15). Under the T1-weighted image mode, cells were exposed to different concetrations of MPS@AuNFs (0.4 mM to 0.6 mM). As shown in the image, 0.45 mM or higher concentration is sufficient enough to record the bright image in MRI. 3.6. Role of Different-Shaped AuNPs in Drug Release. 3.6.1. Temperature-Triggered. In vitro drug release behavior of MTX from AuNPs modified MPS was investigated using a dialysis membrane in the PBS medium at three different temperatures: (1) 25 °C (room temperature), (2) at 37 °C (body temperature), and (3) at 42 °C (LCST) (Figure 4A, B). From the figure, it is clearly visualized that at body temperature, no significant drug release was observed from any one of the polymersome or MPS, which shows that prepared polymersome/MPS were free from problem of drug leakage at normal temperature or body temperature. The maximum drug release of ∼70−80% was obtained at 42 °C within 5 min of time duration in different types of polymersome/MPS. Thus, the drug release profile confirms that the release rate is dependent on temperature and after a certain temperature, the rate of drug release become passive and smooth. 3.6.2. NIR-Responsive. Figure 4C shows the effect of NIR radiation on the drug release profile of different-shaped AuNPmodified MPS recorded in PBS medium of pH 7.4 (25 °C), in the absence and presence of NIR light (power density = 1−2.0 W/cm2), at the certain drug release time intervals. In the absence of NIR radiation, there is no release of MTX from MPS, i.e., any effect of shape on drug release was not observed (Figure 4C and Figure S16). However, after NIR irradiation, a sharp increase in drug release was observed in MPS. It was also observed that drug release from MPS depends upon power of NIR radiation also. As shown in the Figure 4D, at the power density of 1.0 W/cm2, the release rate of MTX around 73%, which significantly get increased to 98%, when the power density of NIR source has been increased to 2.0 W/cm2. The power density dependent release could be explained by the photothermal response of AuNPs embedded with MPS. Because of the NIR light irradiation, the AuNPs embedded in MPS produce local heat by the efficient photothermal conversion, which weaken the drug carrier-MTX interactions and increase the mobility of MTX at elevated temperatures. However, when the NIR irradiation was turned off, the AuNPembedded MPS could not produce any local heat effect. Thus, no releasing effect was observed in absence of NIR. According to the literature, the photothermal behavior of AuNPs depend upon their shape or size.29 A similar response was also observed in the different-shaped AuNPs-modified MPS and the drug release in first 5 min is found in the given order: polymersome (0%) < AuNSs@MPS (62%) < AuNTs@MPS (73%) < AuNRs@MPS (83%) < AuNFs@MPS (98%). The flowershaped AuNPs-modified MPS showed the best performance among their different-shaped colleagues. The superior performance of flower shape over triangledue-, rod-, and sphericalshaped AuNPs could be explained on the presence of highest LSPR band around 500−700 nm the presence of sharp-edgelike structure of AuNPs, which significantly increase the surface area and amplitude of the plasmon field. This is the first study of their own kind, where nanoparticle shape-based drug release study was performed and found that spherical NPs are not only the option in the field of drug delivery or biomedical studies. K


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Figure 5. (A) Cell viability study of MTX and MTX-loaded MPS in the presence of NIR radiation. (B) Blood clearance study of AuNFs@MPS. Confocal microscopic images showing cellular uptake of AuNFs@MPS-without folic acid after different time intervals: (C, D) 0 and (E, F) 3 h. The bar in the images is 25 μm.

3.7. Role of Shape of AuNPs in Cytocompatibility. It is very important to investigate the toxicity of MPS before employing them as a drug delivery system. To examine this, we examined the cell viability of MCF-7 cancer cells in the presence of polymersome without MTX. When the cells were incubated with 0.0, 50.0, and 100.0 mg L−1 of MPS/ polymersome for 24 h, all the prepared MPS/polymersome showed a maximum decrease in cell viability of about

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