Colloidal micelles of block copolymers as nanoreactors, templates for gold nanoparticles, and vehicles for biomedical applications.

Colloidal Micelles of Block Copolymers as Nanoreactors, Templates for Gold Nanoparticles, and Vehicles for Biomedical Applications Mandeep Singh Bakshi PII: DOI: Reference:

S0001-8686(14)00243-7 doi: 10.1016/j.cis.2014.08.001 CIS 1470

To appear in:

Advances in Colloid and Interface Science

Received date: Revised date: Accepted date:

17 April 2014 29 July 2014 7 August 2014

Please cite this article as: Bakshi Mandeep Singh, Colloidal Micelles of Block Copolymers as Nanoreactors, Templates for Gold Nanoparticles, and Vehicles for Biomedical Applications, Advances in Colloid and Interface Science (2014), doi: 10.1016/j.cis.2014.08.001

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ACCEPTED MANUSCRIPT 1

Colloidal Micelles of Block Copolymers as Nanoreactors, Templates for Gold

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Nanoparticles, and Vehicles for Biomedical Applications Mandeep Singh Bakshi

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Department of Chemistry, Wilfrid Laurier University, Science Building, 75 University Ave. W.,

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Waterloo ON N2L 3C5, Canada.

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Abstract

Target drug delivery methodology is becoming increasingly important to overcome the shortcomings of conventional drug delivery absorption method. It improves the action time with

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uniform distribution and poses minimum side effects, but is usually difficult to design to achieve the desire results. Economically favorable, environmental friendly, multifunctional, and easy to

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design, hybrid nanomaterials composed of colloidal micelles of amphiphilic block copolymers

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and gold (Au) nanoparticles (NPs) have demonstrated their enormous potential as target drug delivery vehicles. This account summarizes their synthesis, characteristic features, and important

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biological applications.

Block copolymers possess inherent ability to self-aggregate in well-defined micelles which show excellent ability as nanoreactors to synthesize Au NPs in vitro. Au NPs use the micelles as soft templates to self-assemble which eventually generate hybrid morphologies on the basis of their mutual symbiotic relationship. Since the micelle is a pseudo-fluid phase, therefore it responds to external stimuli such as temperature, pH, and ionic strength, and hence, proves to be fine vehicle for drug release; while the presence of Au NPs simultaneously act as contrast agents to mark the target. A combination of both (micelle and NPs) makes them fine target delivery vehicles in variety of biological applications where precision is primarily required to achieve the desire results as in the case of cytotoxicity of cancer cells, chemotherapy, and computed tomography guided radiation therapy. We systematically demonstrate the potential of such systems as target delivery vehicles by taking a comprehensive view point for different kinds of block copolymer – Au NPs hybrid systems in terms of their physiochemical aspects and functionalities.


Key words: Block copolymer micelles, gold nanoparticles, nanoreactors, biomedical applications.

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Introduction

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Water soluble non-ionic block copolymers demonstrate potential industrial applications due to their unique architecture of three blocks (triblock copolymers, TBPs) arranged in an

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alternative arrangement of polyethylene (PEO) and polypropylene (PPO) oxides units as in the case of pluronics1-6. A TBP becomes predominantly hydrophilic when number of PEO units

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exceeds the PPO units, and on the other hand, acquires a predominant hydrophobic nature when reverse happens7-9. Micelle formation is also triggered by the temperature variation1-6 which

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induces greater dehydration to PPO in comparison to PEO units that in turn brings predominantly

Core

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Corona

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hydrophobic PPO units to aggregate in the core of the micelle while predominantly hydrophilic

Guest molecule

Micelle Surface cavity

Fig 1. Schematic representation of a TBP micelle showing core and corona. Core is mainly made up of PPO and corona by PEO units. Arrangement of PEO units leads to the formation of surface cavities to accommodate guest molecules. (from ref 26) PEO arrange themselves in the corona (Fig 1). The temperature where the micelle formation occurs is known as the critical micelle temperature (CMT)10-13. Further heating beyond CMT, drastically reduces the hydration of the micelles and thus induces cloudiness which is known as the cloud point (Cp)14-16. TBP micelle demonstrates interesting shape transitions which are closely associated with number of factors such as the molar masses of PEO or PPO units, salt additives, nature of the solvent, concentration, and temperature.17-19 Usually, micelles with a greater number of PEO units than PPO are tend to form compound micelles7,20-24 where several small micelles exist in large groups, but predominantly hydrophobic micelles are mainly well defined and do not readily aggregate into compound micelles.7


Since the TBP micelles are usually very large (few to several hundred of nm) in comparison to the micelles of conventional monomeric surfactants, therefore, they undergo

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several structure transitions (i.e. spherical micelles  thread like micelles  vesicles etc.) with concentration as well as temperature variations.17-19 These micelles are lined with surface

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cavities25-27 (Fig 1) which are formed by the surface arrangement of PEO units and are in direct

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contact with the aqueous phase to constitute the micelle – solution interface. Temperature induced dehydration of the micelles allows the compact arrangement of the surface cavities which in turn generates micelles of larger aggregation number with a greater number of surface

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cavities. Surface cavities are embedded with electron donating ether oxygens and hence readily participate in the redox reactions.25-27 As one polymer molecule is mainly contributing towards

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the formation of one surface cavity, therefore, it accepts one guest ion (oxidizing agent) per cavity where the host – guest fit is very much related to the size of the PEO block (Fig 1).26,27 A

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smaller cavity produced by few PEO units (i.e. a smaller PEO block) is unable to properly

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accommodate a guest molecule. In contrast, a larger cavity (i.e. a larger PEO block) will form a large bucket quite big enough to accommodate a guest molecule. Hence, reducing ability of a

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surface cavity is also very much related to its size. Several surface cavities present on the surface of a micelle can simultaneously reduce almost equivalent number of oxidizing agents if all oxidizing agents can be accommodated in the cavities. In addition, micelle environment is also a contributing factor for a proper host – guest fit. Fully hydrated surface cavities with low aggregation number at low temperature may not accept as many oxidizing agents as many accepted by the partially hydrated or dehydrated cavities with higher aggregation number at high temperature.26

Complex gold ions (AuClO4-) act as a fine oxidizing agents and interact with the surface cavities to get reduce from Au(III) into Au(0), and hence initiate the surface redox reaction to produce gold nanoparticles (Au NPs). First, tiny nucleating centres are created which grow on the surface cavities and eventually lead to the formation of Au NPs.25-27 Nucleation can also occur between the nucleating centres occupying the adjoining surface cavities or through autocatalytic thermodynamically controlled reduction. In this way, micelle environment, shape, and size of the micellar assemblies act as soft-templates which govern the overall morphology of NPs.26,27 Small micelles with few surface cavities cannot affectively lead the nucleating centres


to well defined morphologies in comparison to large micelles with several surface cavities. Likewise, compact arrangement of surface cavities allows nucleating centres to self-nucleate and

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allows NPs to replicate the shape and size of the micelle. Such micelle-NPs hybrid assemblies can also be used for various applications such as catalysis, drug delivery vehicles, and

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cytotoxicity.28 This review accounts for various aspects of block copolymer micelles based on

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their architecture and amphiphilic behavior to design appropriate hybrid materials best suited for such applications. A comprehensive view of the synthesis, stability, and characteristic features of several water soluble and insoluble block copolymers – Au NPs hybrid systems have been

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discussed along with their unique biological applications.

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Micelles (structured reducing agents) as nanoreactors

In order to understand the synthesis of Au NPs by using the micelles of block

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copolymers, we need to ensure the presence of micellar phase along with the gold salt. The

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micelle formation of block copolymers is similar to that of the conventional surfactants where predominant amount of the polymer exists only in the micellar phase and is in thermodynamic

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equilibrium with less predominant monomeric form. Water soluble block copolymers such as pluronics and tetronics generate well defined micelles lined with surface cavities at water – solution interface. When such micelles come in contact with the gold ions, they electrostatically interact with them through surface cavities to initiate the reduction reaction. Thus, a typical synthesis of Au NPs can be best studied in a ternary combination of block copolymer + gold salt + water in the absence of any other reducing agent.7,26,27 It is to be mentioned that the monomeric form of block copolymer does not effectively involved in the reduction reaction due to the lack of surface cavities and hence reduction is primarily initiated by the micelles only. The reduction potential is further related to the number of surface cavities or the concentration of the micellar phase. Such type of reducing agent can be termed as structured reducing agents where structural factors are mainly directing the reduction. In other words, monomeric form is not a structured reducing agent because it cannot create surface cavities and hence cannot participate in the reduction reaction.26,27 Thus, the formation of the structured reducing agent is an important step to develop efficient nanoreactors for the synthesis of Au NPs. Apart from such block copolymers, micelles of other copolymers like polystyrene-block-poly(4-vinylpyridine),


poly[tert-butylstyrene-b-sodium (sulfamate/carboxylate-isoprene)], and poly(N-isopropylacrylamide)-b-poly(1-(30-aminopropyl)-4-acrylamido-1,2,3-triazole hydrochloride),28-31 have also

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been employed as nanoreactors for the synthesis of Au NPs. Although, these systems possess their own importance from technical point of view, they usually do not lead to well defined

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micelles – Au NPs hybrid morphologies. The micelle formation is basically an aqueous phase

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process where hydrophobic interactions drive the formation of well-defined morphologies which are usually rarely generated in the non-aqueous phase. In addition, for any possible biomedical applications, their stability in the aqueous phase is primarily required where they can be used as

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efficient drug release vehicles. Various techniques can be employed for the determination of the micelle formation of block copolymer micelles, and among them, the most common are the

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surface tension, fluorescence, and UV measurements which are highly sensitive toward the

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aggregation behavior of block copolymers.

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Fig 2. (a) UV-visible scans of L31 (2 mM) + HAuCl4 (0.25 mM) + water ternary mixture with time at 70 oC. Black arrows represent three peaks at 220 nm, 290 nm, and 550 nm due to AuCl4ions, LMCT complex, and surface plasmon resonance of Au NPs, respectively. (b) Intensity versus time plots of these peaks. (c) Plots of intensity at 290 nm and 550 nm versus temperature


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for various ternary mixtures with different concentrations of L31. Vertical arrows indicate the CMT region. (d) A variation in the intensity of methyl orange at 460 nm versus temperature for ternary mixtures with different concentrations of L31 and without the presence of HAuCl4. (from ref 26)

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Here, one such typical reaction is shown by following the reaction kinetics with the help of UV-visible studies. The UV-visible studies are the best spectroscopic technique which can be

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employed with great ease not only to determine the micelle formation of block copolymer but also to simultaneously monitor the synthesis of Au NPs on the basis of their surface plasmon

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resonance (SPR).26,27 A typical reaction of L31 (PEO2-PPO16-PEO2)+HAuCl4+water mixture is depicted in Fig 2a with three peaks at 220, 290, and 550 nm due to AuCl4- ions, ligand to metal

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charge transfer band (LMCT), and SPR32-36 of Au NPs. A variation in these peaks (Fig 2b) explains the reaction mechanism that how reduction is initiated to generate nucleating centres, which eventually convert into NPs. As reduction initiates, it consumes AuCl4- ions (220 nm

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peak) through LMCT complex (peak 290 nm, Fig 2c) and produce Au NPs (550 nm peak). In

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this process, every surface cavity of L31 micelle produces approximately one Au atom and the number of surface cavities is actually related to the aggregation number of the micelle which

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further depends on the hydrophobicity and increases with the number of PPO units. Small micelles with few surface cavities are only able to trigger the nucleation when they undergo inter-micelle collisions under diffusion controlled process.25 In order to confirm that the reduction reaction is actually initiated by the micelles, parallel micelle formation in the absence of Au NPs needs to study. This is best studied by following the CMT of a TBP. Temperature causes a significant dehydration in the TBP resulting in the micelle formation. We can determine the CMT of a TBP simply by taking a UV-visible absorbing dye such as methyl orange (MO) and follow its absorption. Because of the ionic nature of MO, it electrostatically interacts with the polymer. However, as temperature increases, polymer aggregates and hence releases the MO in the solution and thus its absorbance increases indicating the onset of micelle formation (Fig 2d). The temperature where it happens is known as CMT. Likewise, the consumption of LMCT (at 290 nm, in Fig 2c) exactly traces the CMT behavior to convert into Au NPs, suggesting the fact that the reduction of Au(III) into Au(0) is completely controlled by the micellar assemblies. This is true for all pluronics but CMT varies with respect to the hydrophilic/hydrophobic ratio (PEO/PPO). Increase in this ratio increases the hydration of micelles and hence increases the


CMT and vice versa. Thus, the synthesis of Au NPs only follows the CMT of any pluronic used and remarkably only related to the single parameter i.e. CMT. In this way, wherever pluronics

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are required as nanoreactors for the material synthesis, one needs to just control the CMT. It works well as far as the synthesis of Au NPs concerned, but the synthesis of other noble metal

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NPs such Ag and Pt can also be achieved in a similar manner. Nevertheless, synthesis of Au NPs

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is always much easier because of its higher positive standard reduction potential in comparison to Ag and Pt, and that is why even weak reducing agents like block copolymers can easily reduce the Au(III) into Au(0). After understanding the physiochemical aspects of block copolymer

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micelles and their nanoreactor behavior in the synthesis of Au NPs, we can focus on the shape

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and size their self-assembled morphologies to understand the soft template effect. Shape and size factors of nanoreactor micelles

The structural aspects of block copolymer micelles and their reaction sites in terms of

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―nanoreactors‖37-40 cannot be precisely evaluated from the conventional physiochemical

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techniques, because these studies only indicate their solution phase behavior without pinpointing the nature of association between the micelles and Au NPs. Since, micelles are considered to be a

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pseudo-phase thermodynamically different from the monomeric phase of a conventional


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Fig. 3. (a) A TEM micrograph of F68 micelles loaded with small NPs synthesized in a HAuCl4 (0.5 mM) + F68 (5 mM) + water ternary reaction at a precise temperature control of 40±0.1 oC. White arrows indicate the presence of distinct NPs of different sizes. (b) and (c), respective single compound micelles with NPs at different locations. (d) and (e) compound micelles loaded with NPs prepared with 10 mM F68. (f) A SEM micrograph of much larger P103 micelles loaded with well defined distinct Au NPs synthesized in a HAuCl4 (2 mM) + P103 (10 mM) + water ternary reaction at a precise temperature control of 40±0.1 oC. (g) A high resolution SEM image showing a single micelle loaded with NPs. (from ref 27) surfactant, its association with Au NPs in the case of pluronics allows it to be completely extracted in the dried state and visualized through imaging techniques like microscopic studies (i.e. TEM and SEM) enabling us to precisely quantify the shape and size of such assemblies. This is an important aspect to be explored in view of their biological applications where such assemblies are employed for different functionalities ranging from bio-markers to drug delivery vehicles in biological systems. Hence, their shape and size is an important factor to determine in order to trace the mechanism behind the cell internalization and subsequent applications. The symbiotic relationship between the micelles and Au NPs arises from the participation of micelle surface cavities in the synthesis of NPs and in return, NPs adsorb on the micelle surface to


achieve colloidal stabilization. TEM studies demonstrate that how such relationship result in soft template effect where small NPs are precisely arranged on the surface of micelles. This is

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illustrated in Fig 3a-c where well defined spherical compound micelles or vesicular aggregates27 of 27±0.7 nm of predominantly hydrophilic F68 (PEO78-PPO30-PEO78) carry dark NPs of 2 – 3

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nm on their surfaces (indicated by white arrows). No independent NPs are observed which

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suggests that NPs grow on the surface cavities and use micelle surface as template to selfassemble. NPs grow in size and become more prominent when larger amount of F68 is used (Fig 3d,e) that in turn increases the number of surface cavities and leads to the greater number of

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nucleating centres. However, the micelles grow in size if a predominantly hydrophobic TBP such as P103 (PEO17-PPO60-PEO17) (Fig 3f) is used. A much bigger PPO block promotes the

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hydrophobic interactions among the polymer molecules resulting in an increase in the aggregation number, and hence, their ability to carry even larger NPs also increases (Fig 3g).

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NPs synthesized in this way are often close to monodisperse because their growth is mainly

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controlled by the number of nucleating centres produced. However, we cannot equate the size of each NP with the possible size of the surface cavity since every growing nucleating centre in

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aqueous phase has the ability to promote the autocatalytic33,34 conversion of Au(III) into Au(0) on its surface, and it continues as long as the supply of precursor continues. That is the reason why the size of NPs of Fig 3g and f is much bigger than that of Fig 3b-e. In addition, predominantly hydrophobic micelles are always much more compact due to less hydration in comparison to predominantly less hydrophobic micelles. Compact micelle obviously generates compact arrangement of surface cavities7,27 and hence promotes the ability of growing nucleating centres to merge with each other. This factor is also considered to be responsible for the increase in the size of NPs. Therefore, it is possible to control the size of NPs by simply controlling environment of the micelle. It is important to note that the NPs produced by the surface cavities of micelles are roughly polyhedral and monodisperse which is usually an important factor for their possible role as catalysts41-44 to increase surface area. Micelles of water – insoluble Pluronics and synthesis of Au NPs This part explains the role of micelles of aqueous insoluble TBP in the synthesis of Au NPs. This happens when a PPO block becomes extraordinarily larger than the PEO block, it renders TBP water insoluble due to its predominant hydrophobicity. L121 (PEO5-PPO68-PEO5)


is one of the best example of such a strongly hydrophobic TBP. It can be made water soluble if sufficient hydrophilicity is introduced by allowing the ether oxygens to get hydrated. This can be

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done by incorporating conventional surfactant monomers in the micelles of such kind of polymers to generate mixed micelles.10-16 The mixed micelles thus created are usually well

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defined and relatively less polydisperse because of the existing synergistic interactions between

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the monomeric forms of surfactant and TBP which are basically the driving force for the mixed micelles formation. The less polydisperse behavior obviously leads to a better template with uniform shape and size for accommodating Au NPs. However, the presence of a conventional

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surfactant in the mixed micelle has a dramatic influence on the arrangement of surface cavities of TBP thereby affecting the reduction potential of the mixed micelle. Several studies of the mixed

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micelle behavior of ionic surfactants like sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium chloride (CTAC) with a relatively less hydrophobic P123 (PEO20-PPO70-PEO20)

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suggest the disruption of polymer aggregates due to the induction of conventional surfactant

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monomers which bring significant hydration to the PPO block.45 Low concentrations of ionic surfactants mainly form polymer rich mixed micelles while intermediate concentrations

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simultaneously produce small surfactant – rich aggregates.46 Similar interactions were reported with nonionic surfactants but with relatively of lesser magnitude than ionic surfactants based on the regular solution approximations.47 Therefore, choice of an appropriate surfactant is an important aspect to generate well defined stable micellar templates suitable for synthesizing as well as accommodating Au NPs. Zwitterionic surfactant7 of low hydrophobicity (i.e 3-N,Ndimethyldodecyl ammoniopropane sulfonate, DPS) in a relatively low amount is always best suited for the stable mixed micelle formation for their subsequent use in the synthesis of Au NPs (Fig 4a) in comparison to ionic surfactants. In addition, these mixed micelles are highly temperature sensitive because of their predominantly hydrophobic nature and hence undergo dramatic dehydration through distinct phase changes which depict marked influence on the synthesis of Au NPs. This is demonstrated by different regions of a phase diagram in Fig 4b. In the first region, only LMCT complex exists within a certain temperature range and hence no absorbance due to SPR of NPs is observed. This region dramatically shrinks to low temperature range with a decrease in the mole ratio of DPS/L121 because the mixed micelles are


predominantly hydrophobic in nature due to a smaller amount of zwitterionic surfactant. It also

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indicates that the low mole ratio does not promote the LMCT complex formation because the

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Fig 4. (a) Plot of absorbance versus wavelength for DPS+L121+HAuCl4 with DPS/L121 mole ratio = 2.5 at different temperatures from 10 – 70 oC. Black arrow indicates the absorbance due to surface plasmon resonance of Au NPs. (b) Intensity versus temperature plot for different mole ratios of DPS/L121. Dotted arrows indicate the nucleation temperature (NT). (c) Intensity versus temperature plot of methyl orange at different mole ratios of DPS/L121. (d) Plot of NT versus surfactant/L121 mole ratios for DPS, TPS, and HPS. (from ref 7) reduction is primarily carried out by the TBP surface cavities whose number is much greater in the presence of little amount of surfactant. Part II starts soon after the formation of nucleating centres and depicts their growth with temperature. The temperature where the synthesis of nucleating centres becomes prominent is called the nucleation temperature (NT) (see Fig 4b). As nucleating centres convert into tiny NPs, their absorbance shows a sudden increase but remains weak over a wide temperature range. When the temperature reaches the part III, a dramatic rise in the intensity indicates a pronounced growth in the number density of NPs due to inter – particles fusions as substantial dehydration of mixed micelles occurs. Parallel control


experiments in the absence of HAuCl4 and in the presence of methyl orange dye show similar behavior (Fig 4c). This demonstrates that the nucleation is closely controlled by the micelle

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surface cavities even in the mixed micelles of water insoluble pluronics and hence the growth kinetics follows the micellar transitions. Since dehydration increases the hydrophobicity and

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makes micelles more compact, hence, it provides a compact arrangement of surface cavities. It enhances the electron donating ability and provides sufficient energy to convert LMCT into

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nucleating centres to grow into tiny NPs.26 In contrast, nothing like this is demonstrated by Fig 2b where no micelle transitions of water soluble pluronic micelles are observed with the same

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temperature range due to their predominantly hydrophilic nature. A variation in the NT with respect to mole ratio explains the dramatic influence of surfactant on the number of surface

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cavities of aqueous insoluble L121 micelle (Fig 4d). NT decreases with the decrease in the mole ratio or decrease in the amount of surfactant, which means that the higher amount of DPS (mole

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ratio = 2.5) reduces the number of surface cavities in the mixed micelle to a greater extent than

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lower amount (mole ratio = 0.5) and hence delays the nucleation. Likewise, greater hydrophobicity in the order of DPS < 3-N,N-dimethyltetradecyl ammoniopropane sulfonate

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(TPS) < 3-N,N-dimethylhexadecylammonio-propane sulfonate (HPS), also promotes the solubilization of L121 and destabilizes the L121 micelles which reduces the NT systematically in the order of DPS > TPS > HPS. All curves merge with each other at low mole ratio and extrapolate to 10 oC in the absence of surfactant which means that L121 is capable of generating nucleating centres as soon as AuCl4- ions are accommodated in the surface cavities. In other words, the micelles of water insoluble L121 act as much stronger reducing agents and better nanoreactors than the micelles of water soluble pluronics due to lesser hydration, that promotes stronger reducing conditions for the synthesis of Au NPs. Structural aspects of water insoluble micelles It is interesting to discuss that how micelles of water insoluble L121 in the presence of surfactant are different from that of previously discussed water soluble block copolymers. TEM images reveal that these micelles are well defined and loaded with tiny NPs. Each micelle contains uniformly distributed Au NPs of 2 – 4 nm which are thoroughly embedded in the


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Fig 5. (a) Low resolution TEM image of a sample prepared with DPS/L121 mole ratio = 0.5 at 70 oC. Dotted white circles enclose single micelle. (b) and (c), high resolution images of different roughly spherical micelles with self assembled Au NPs of 2 – 6 nm in size. White arrows in (c) indicate some fused NPs. (d) and (e), high resolution TEM images of micelles of different sizes along with sub-divided micelles bearing small NPs. (f) Low resolution TEM image of flower like large compound micelles prepared with DPS/L121 mole ratio = 2.5. (g), (h), (i), and (j), high resolution images of the same sample with different magnification showing core – shell morphology as well as tiny Au NPs. (from ref 7) micelles (Fig 5a-c). This is only possible if the number of surface cavities is much greater which is obviously expected from the predominantly hydrophobic nature of these micelles and compact arrangement. However, this kind of arrangement exists only at low mole ratios of surfactant/L121. But as the mole ratio exceeds 1:1, it brings dramatic changes and tend to disintegrate the large micelles into smaller ones (Fig 5d,e). This is all due to greater solubilization power of DPS to solubilize the polymer that results in the formation of smaller mixed micelles. Likewise, greater amount of DPS than L121 with mole ratio = 2.5 causes the formation of much larger (~ 1 m), monodisperse, and roughly spherical flowerlike micelles (Fig 5f). Each morphology is made up of less dense large core and relatively more compact thin shell


of ~ 50 nm (Fig 5g,h). Different micelles are interconnected with each other through the overlapping of the thin shells of adjoining micelles (Fig 5i). The large core predominantly

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contains a significantly higher number density of 2 – 3 nm Au NPs than in the shell (Fig 5j) because much larger amount of DPS has the ability to solubilize predominantly hydrophobic

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PPO block of L121, and hence accommodates their surface cavities in the anterior of the micelle.

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This sequence of images shows some interesting properties of the mixed micelles. One would see that the greater amount of the surfactant in fact induces greater hydration in the L121 micelles and hence their size increases from few hundreds of nm to over micrometer size.

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Likewise, the surface arrangement of surface cavities is also changed. At low DPS concentration, NPs were thoroughly distributed over the whole surface of the micelles, but now they are more

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concentrated only in the anterior of the micelle. Micelle structure transitions

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These micelles undergo significant structure transitions48,49 if the hydrophobicity of the zwitterionic surfactant is enhanced. This is all because of the introduction of the longer

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hydrocarbon chain in the block copolymer micelle to form thermodynamically stable mixed micelles. Micelles of either conventional surfactants or amphiphilic polymers usually attain spherical or roughly spherical shape due to a natural tendency to acquire higher volume to surface ratio. However, this ratio alters when the mixed micelle is formed from monomers of different architecture10,12,16 and hence shape transitions occur to minimize the free energy of selfaggregation. Thus, replacing DPS with TPS produces rectangular (Fig 6a) and hemispherical (Fig 6b) micelles thoroughly loaded with tiny NPs. An oval shaped micelle (Fig 6c) probably is obtained from the bifurcation of the elongated micelle (Fig 6d). However, further increase in the


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Fig 6. (a – d), High resolution images of different shapes of micelles bearing tiny NPs of 2 – 3 nm of a sample with TPS/L121 mole ratio = 0.5. (e) Similar image of long rod shaped micelles bearing tiny NPs a sample with HPS/L121 mole ratio = 0.5. (g) and (h), high resolution TEM image of different micelles in which white block arrows indicate the strands of fused NPs. (from ref 7) length of hydrocarbon chain as of HPS even induces more deformation and many long rod

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shaped micelles are produced apart from other shapes. This sequence of different micelle templates demonstrates a high degree of micelle transitions from sphere to rod and are governed by the increasing hydrophobicity in the order of DPS < TPS < HPS. In addition, any amount of zwitterionic surfactant greater than that of L121 does not yield any micelle templates, rather produces independent NPs of ~100 nm with predominant plate-like50 triangular and hexagonal shapes. Thus, low amount of low hydrophobic conventional surfactant is the best choice to produce well defined spherical micelles of a water insoluble pluronic for the accommodation of Au NPs. Since the reduction of Au(III) to Au(0) is precisely controlled by the surface cavities in aqueous phase, therefore, minimum possible amount of the conventional surfactant should be used so as to have little effect on the overall shape and size of the TBP micelles and hence the micelle retains its predominant hydrophobic nature. One can find a marked difference between the overall shape and size of the micelles of predominantly hydrophobic L121 (summarized in Fig 7) from that of predominantly hydrophilic


L121

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Fig 7. Schematic representation of the mixed micelle formation between L121 and zwitterionic surfactants and their subsequent use as micelle templates for the self assembled Au NPs. Top reaction shows the formation of spherical L121+DPS mixed micelles in the L121 rich region of the mixture. Middle reaction shows the DPS rich region. Lower reaction shows that by using TPS or HPS instead of DPS induce their longer hydrocarbon tails in the L121 micelles thus causing structure transitions.(from ref 7) F68 (Fig 3) block copolymer. Hydrophobic micelles are well defined, large, and carry several tiny NPs while hydrophilic micelles are much smaller without proper shape and contain relatively less number of large NPs at comparable concentrations of both pluronic as well as gold salt. Therefore, predominantly hydrophobic block copolymers are the better nanoreactors for stable Au NPs – micelle self-assembled morphologies.


Micelles of star shaped block copolymers “Tetronics” Tetronics belong to a category of star shaped block copolymers.1,51-56 They are basically

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homologues of pluronics but exist in a star shaped structure with four arms connected to a central diamine functional group (Fig 8). Their self-assembled behavior is highly temperature as well as

HO(OE)15(OP)17

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pH sensitive.1,56 Low pH turns the amine moiety into ammonium groups and macromolecule (PO)17(EO)15OH N-CH2-CH2-N

(PO)17(EO)15OH

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Fig 8. Structural formula of star shaped T904 and its schematic representation. (from ref 56)

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acquires a net positive charge which restricts its aggregation. High pH allows it to attain a neutral form which under the effect of concentration and temperature produces micelle like aggregation.

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Because of its unique architecture, the micelle formation in tetronics is much different from that of pluronics because this geometry is obviously not expected to generate a conventional micellar arrangement as observed in pluronics due to steric constrains in four arms of PPO/PEO blocks which leads to a highly hydrated micelles. Like pluronics, they also produce in vitro Au NPs by reducing Au(III) into Au(0) by employing their surface cavities (Fig 9a). This reaction is highly affected by the change in medium pH because of the central amine moiety (Fig 9b). High pH, where amine moiety is available for the electron donation to the electropositive metal centre of gold ions, induces instant reduction, while the low pH requires high temperature to do so. Low pH protonates the amine moiety and brings excessive hydration. Hence, the synthesis of Au NPs is significantly affected by both temperature as well as pH variations. In order to initiate the reduction by the surface cavities, excessive hydration needs to be reduced to allow the surface cavities to interact with the gold ions. Thus, nucleation is carried out at elevated temperature at low pH rather than at the high pH. The temperature where the nucleation becomes prominent, is the NT (see arrows in Fig 9b) just like that of Fig 4b for pluronics and its variation for three tetronics [i.e. T904 (EO = 15, PO = 17) average molar mass = 6700, T908 (EO = 114, PO = 21)


average molar mass = 25,000, and T1307 (EO = 72, PO = 32) average molar mass = 18,000] is

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Fig 9. (a) A typical example of a reaction in aqueous phase of 10 mM T904 with 0.5 mM HAuCl 4 at pH = 6 under the effect of temperature variation from 20 – 70 oC and simultaneously monitored by the UV-Visible measurements. Red dotted line refers to blank (i.e. aqueous 10 mM T904 without gold salt) and blue dotted line represents the scan at 70 oC. Scans from bottom to top follow 20 – 70 oC temperature range. (b) Shows the variation of intensity of 540 nm peak versus temperature for the same reaction at different pH. NT represents the nucleation temperature from where the synthesis of gold nucleating centres starts. (c) Plot of NT versus pH for the reactions of different tetronics. (d) UV-Visible scans of aqueous 10 mM T904 in the absence of HAuCl4 and presence of MO at pH = 6 under the effect of temperature variation from


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20 – 70 oC. Arrow point to the blue shift in the absorbance with single isobestic point. (e) A comparative plot of the variation in the wavelength of absorbance of MO of (d) and intensity of Au NPs absorbance from (a) versus temperature. (f) Gel electrophoresis of all samples prepared in (b) at different pH. (from ref 56)

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for T904 followed by T1307 and T908. A strongly basic medium facilitates the instantaneous reduction of Au (III) into Au(0) since all NT values from three curves merge at pH = 12

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irrespective of the nature of tetronics at 20 oC. Here too, NT is closely related to the CMT of tentronics. The CMT can be evaluated from the variation in UV-visible absorbance of MO in the

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absence of gold salt (Fig 9d). A blue shift in the 460 nm absorbance of MO through a single isosbestic point around 440 nm with the rise in temperature indicates the micelles formation. The

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blue shift follows the reverse sigmoidal curve, the centre of which coincides with the forward sigmoidal curve of the intensity of Au NPs (Fig 9e). The centre of the former curve denotes the temperature where isosbestic point appears in Fig 9d and hence represents the micelle formation

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temperature of T904.57,58 Thus, like pluronics, micelles of tetronics are capable of in vitro

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synthesis of Au NPs. Tetronic stabilized Au NPs are charged due to the presence of amine functional groups and hence can be very well evaluated with the help of gel electrophoresis

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which also demonstrates the pH effect (Fig 9f). Structural aspects of tetronic micelles Microscopic evaluation provides direct comparison between the shape and size of the micelles of tetronics with that of pluronics which is obviously expected from their marked difference in the molecular structures. Micelles of tetronics (Fig 10) are much hydrated at low pH and hence they create a pool of water in their vesicular like self-assembled state due to their unique molecular arrangement (Fig 10e,f). Such kind of morphology produces surface cavities at their surface which are directly involved in the synthesis of Au NPs and hence NPs adsorbed on the surface of highly hydrated compound vesicular assemblies (Fig 10c,d). The hydrated pool is usually seen as a transparent area in TEM surrounded by a dark concentric shell in the dried state. This kind of morphology is the most probable way in which star shaped T904 can be arranged because predominantly hydrophobic PPO blocks are sand-witched between predominantly hydrophilic PEO blocks from the outside and amine groups from the inside of the T904 macromolecule. However, high pH mainly produces compact aggregates with no clear


distinction between the core and the shell regions because it prevents the protonation of amine groups and hence reduces the hydration that in turn dehydrates the micelles. The proposed

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hydrated structure is supported from the energy minimization59,60 studies by scanning the torsion angles of side chains to relieve steric interactions and to seek a position in which there is no van

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der Waal’s contact between either side of the bond with a convergence criterion of less than 0.01

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kcal/mol. The aggregated form produces the stacking arrangement among different monomers to generate a micelle like assembly where PPO along with the diamine moiety constitutes the core. This stacking arrangement allows amine groups of different monomers to remain in the vicinity

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of each other while generating a similar arrangement among EO and PO. This type of

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Fig 10. (a) TEM image showing large patches of vesicular assemblies of T904 along with Au NPs as dark dots of sample of Fig 2a. Small black dots of (a), close up in (b), and high resolution in (c) are fine spherical shaped Au NPs of 17±5 nm. (d) Shows two side wise fused vesicular assemblies bearing groups of small Au NPs while (e) is the high resolution image showing the presence of Au NPs mainly on the surface of core – shell type vesicle. (f) Shows several core –


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shell type vesicles fused together in a group bearing Au NPs mainly in the shell region of each vesicle. (g) and (h) Show the corresponding schematic representation of core – shell type vesicle and the alignment of P904 macromolecules based on energy minimization. (from ref 56) Thus, tetronic micelles are considered to be more suitable for their pronounced pH and

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temperature responsive behaviors in comparison to pluronic micelles because of their inherent hydration induced by the unique architecture. This makes them good candidates for vehicles for

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drug release under different temperature and pH conditions and more specifically when a formulation is made at room temperature and neutral pH can effectively release a drug

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physiological temperature (37 oC) and even at low pH as in the digestive track.

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Thiol-substituted block copolymer micelles and Au NPs Above studies were specifically related to the block copolymer micelles as nanoreactors without thiol moiety for the synthesis and self-assembled behavior of Au NPs. Since, the block

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copolymer micelles were without thiol moiety, therefore, no covalent association was possible

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between the micelles and the Au NPs. Nevertheless, the involvement of surface cavities in the synthesis of Au NPs produced quite stable micelles bearing Au NPs (Fig 7). Likewise, another

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strategy can be used to self-assemble Au NPs by choosing thiol substituted block copolymer


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Fig 11. Procedure of pluronic micelle shells cross-linked by Au NPs. Synthetic Scheme of nanocapsules from Au NPs stabilized pluronic micelles. (Reproduced with permission from ref 63) micelles where NPs are allowed to self-assemble under the effect of covalent interactions with

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thiol moieties.61-63 Such micelles are in fact act as good soft templates rather than nanoreactors because they are not involved in the in vitro synthesis of Au NPs. In other words, pre-

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synthesized Au NPs can be covalently accommodated on the surface of such micelles where micelle – solution interface is lined with thiol terminated functional groups which control the

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self-aggregation of tiny Au NPs as in the case of thiol substituted F127 micelles (Fig 11). Because the core is made up of highly hydrated F127 (PEO97-PPO69-PEO97), therefore, the

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micelles show pronounced thermo responsive behavior which undergo thiol-exchange reaction with glutathione and their morphology spontaneously evolve and reassemble into large ―vesicular‖-like nanocapsules.63 Increasing the temperature causes the collapse of this structure

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and the number-averaged hydrodynamic diameter decreases leading to a reversible behavior (Fig

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11). This allows to use such temperature responsive self-assembled nanomaterials for transfection vectors, DNA-binding agents, protein inhibitors, spectroscopic markers,64-72 and

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place-exchanging reaction.73,74

Monolayer films of block copolymer micelles and NPs In addition to the bulk behavior, block copolymer micelles – Au NPs self-assemble in the monolayers. Usually water insoluble amphiphilic block copolymers are used for monolayer adsorption because water soluble block copolymers show relatively low surface activity.2,3 There are quite a few studies related to the self-assembled behavior of diblock copolymers in monolayers75-78 along with Au NPs. The dodecanethiol-protected Au NPs are physically incorporated around hexagonally ordered micelles of the monolayer film79-81 prepared by the polystyrene block-poly(4-vinylpyridine), PS-PVP, micelles. Iron oxide nanoparticles are then synthesized chemically in the core area of the ordered micelles, resulting in iron oxide nanoparticles surrounded by Au NPs (Fig 12).82 The arrangement of Au NPs is achieved by coating the dodecanethiol groups on the particle surface which results in a hierarchical selfassembly of nanoparticles in copolymer micelles. Although the number of Au NPs encircling each micelle is fluctuated in the film, the total amount around the micelles is controllable with


their concentration in the solution. The methodology demonstrates a good example of how

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different types of functional nanometer-sized building blocks can be organized in specific

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Fig 12. (a) TEM image of an array of iron oxide nanoparticles surrounded by gold nanoparticles; (b) EDX spectra on gold and iron nanoparticles marked by circles and a cross in the enlarged TEM image, respectively. (Reproduced with permission from ref 82)

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arrangements by physical and chemical self-assembling procedures on structured templates

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which can provide controlled fabrication of nanometer-sized building blocks with unique and useful electronic, optical, and magnetic properties. Since different kinds of NPs exhibit their individual unique properties, thus such films can be employed for a variety of applications in optoelectronic, medical, and catalytic fields. Morphology transformation of hybrid micelles and NPs It is equally important to point out the shortcomings of pre-synthesized Au NPs on the overall shape and size of the block copolymer micelles which may induce polydisperse behavior normally not required when such systems are employed for useful applications. Added effect of Au NPs on the aggregated morphologies of diblock copolymers leads to the formation of micelles-Au NPs hybrid assemblies with significant structure transitions (Fig 13). The aggregate morphology changes from vesicles to a mixture of spheres and cylinders by increasing the particle radius and particle volume fraction. It is well exhibited by rod−coil block copolymers self-assembly behaviors because the rod blocks prefer to take ordered packing.83-86 Thus, it is expected that the change of the local ordered packing of rod blocks should greatly influence the self-assembly behavior of the rod−coil copolymers in the presence of NPs. This is also true in


homopolymer liquid crystal systems, where ordered packing of rigid polymer chains can be destroyed by introducing small portion of nanoparticles. Poly(γ-benzyl-L-glutamate)-block-

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poly(ethylene glycol) (PBLG-b-PEG) block copolymer self-assembles into long cylindrical

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Fig 13. Micelles self-assembled from rod−coil block copolymer/nanoparticles mixture in dilute solution. The mass percent of nanoparticles is (a) 0.62, (b) 1.48, (c) 1.85, and (d) 2.47 wt %. The green and red lines are assigned to rod and coil blocks, respectively, whereas the orange particles denote nanoparticles. (Reproduced with permission from ref 87) micelles. By introducing Au NPs, the formed aggregate morphology transforms to spherical micelles due to the breakage of ordered packing of PBLG rods in micelle core, while NPs are adsorbed near the core/shell interface as well as in the core of the micelles.87 This shows that how the addition of NPs influence the self-assemble behavior of the rod−coil PBLG-b-PEG block copolymer in solution and provides useful guidance for designing hybrid polymeric composites with definite microstructures. Therefore, care should be taken in order to achieve a desired block polymer micelle – Au NPs assemblies so that the shape and size of the final morphology is not altered upon the addition of Au NPs. Controlled incorporation of Au NPs in rod/wire shaped micelles Most of the work related to the self-assembled behavior of block copolymer micelles and Au NPs is restricted to spherical or vesicular morphologies where mostly a uniform distribution


of Au NPs is observed due to electrostatic interactions on the micelle – solution interface. However, it is equally important to put the Au NPs in the core of the spherical or rod - like

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micelles88-91 as in the case of polystyrene190-block-poly(acrylic acid)20 (PS190-b-PAA20) copolymer by using PS270-b-PAA15 coated Au NPs92 (Fig 14). The strategy involves the

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formation of the rods in solution in the presence of Au NPs coated with PS270-b-PAA15 diblock

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copolymer which allows about 80% of the Au NPs to be incorporated into the rods. The method

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Fig 14. Block copolymer rods with internalized Au NPs. (Reproduced with permission from ref 92) does not require the block copolymers to bind, solvate, or otherwise interact during preparation with the ions of the metal involved, nor does it involve post assembly chemical reactions within the aggregates, and is thus suitable for a wide range of particles and block copolymer systems since these hybrid aggregates may provide usuful applications in medicine, electronics, and catalysis.93-99 Incorporation of the high dose of Au NPs in block copolymer micellar wires is obviously a challenging task because NPs have pronounced effect on the morphology of the micellar self-assembled structures as discussed previously (Fig 13). However, it can be done by choosing the hybrid micelles where Au NPs are incorporated in the core of a running micellar wire like morphologies (Fig 15) made up of core-shell cylindrical polymer brush of poly (2vinylpyridine) (P2VP) core and polystyrene (PS) shell.100 Block copolymer micelles loaded with Au NPs can be arranged in well-defined structural super-lattices that are difficult to obtain with high density of NPs (Fig 15c,d). The super-lattices assemblies may offer new opportunities in the fields of nanomedicine, high-density data storage, and biosensors; as well as the opportunities for NPs with various functional cores such as magnetic, catalytic, or fluorescence, to produce multifunctional composites materials.101,102 The selective incorporation of encapsulated


monodisperse PS-grafted Au NPs into the PS core of PS-b-poly(4-vinylpyridine) (PS-b-PVP) micelles produce well-ordered hybrid micelles of spherical, cylindrical, or nanosheet

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morphologies.100 The number of NPs in each micelle can be effectively increased by simply increasing the content of NPs and adjusting the ratio of 3-n-pentadecylphenol (PDP) to the P4VP

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units accordingly. The balance between the NPs loading and the PDP addition maintains the

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same micellar morphology while achieving high NPs loading.

Fig 15. Hybrid assemblies formed from PS20K-b-P4VP17K(PDP)2.0 encapsulated of gold NPs (size 7.5 nm) with content of (a) φNP‑M = 14.2%, φNP‑S = 4%, spherical micelles with ∼1 NP; (b) φNP‑M = 33.1%, φNP‑S = 11.3%, spherical micelles with ∼2 or more NPs; (c) φNP‑M = 49.7%, φNP‑S = 20.3%, cylindrical micelles and small amount of spherical micelles; (d) φNP‑M = 56.9%, φNP‑S = 25.4%, dominating nanosheets along with cylindrical micelles. φNP‑M and φNP‑S as the volume fraction of the NPs in the micelles and the volume fraction of NPs in the polymer-based supramolecules, respectively. (Reproduced with permission from ref 100) Biomedical Applications Recent advances in colloidal chemistry and their applications in biomedical field explored several new concepts of target drug delivery systems. Conventional drug methods are almost entirely based on the molecular forms of drugs which disperse in the biological systems via oral ingestion or intravascular injections through the alimentary tract with usually far less


potential to hit the target because the cellular barriers in the form of lipid bilayers are generally

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permeable only to small uncharged solute molecules. However, nanotechnology based colloidal

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Fig 16. (a) Schematic representation of transportation of drugs in the systematic circulation by

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micelles loaded with Au NPs or Au NPs coated block copolymers. micelle loaded with Au NPs; Au NP coated with block copolymer; red discs are the blood cells. (b) Direct injection of the drug loaded micellar assemblies and Au NPs bearing cancer targeting functionalities. Schematic representation of tumor in the form of a yellow ball.

vehicles prove to be promising materials to replace conventional formulations for target delivery biomedical treatments and are expected to reduce the cost with much greater potential to act. Multifunctional surface modification of gold-stabilized NPs by block copolymers Little work has been done in this direction and few reports are available in the literature.103-107 The most interesting aspect of block copolymer coated NPs is that they can be modified with different biological active functionalities. These can be either covalently attached to the polymer back bone or incorporated through the specific interactions. The formulations made from the self-assembled aggregates of polymer and Au NPs for specific drug delivery vehicles can be either injected into the systemic circulation (Fig 16a) if their hemolytic activities are negligible or they can be directly injected to the specific sites as of cancer tumors (Fig 16b). A major objective of nanomedicine is the ability to combine in a controlled manner multiple functions into a single nanoscale entity,108-114 which allows to target particles with great spatial


precision, and hence increases the selectivity and potency of therapeutic drugs. It is possible to have one of the functional group targeting a specific cell type, while another facilitating cellular

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uptake, and the third one directing the particles to a specific subcellular location.

Fig 17. Chemical synthesis of multifunctional gold-stabilized nanoparticles. The structure of block copolymers modified at the apolar PCL end with thioic acid moiety for attachment to a gold core and at the PEO terminus with a functional group for post synthesis modification (a). The chemical synthesis of multifunctional nanoparticles (b). A cartoon of the architecture of the nanopaticles in aqueous solution and the three sequential bio-orthogonal reactions for surface modification (c). (Reproduced with permission from ref 114) Poly(ethylene oxide)-β-poly(ε-caprolactone) (PEO-b-PCL) block copolymers modified at the apolar PCL terminus with thioctic acid and at the polar PEO terminus with an acylhydrazide, amine, or azide moiety have been used as nanocarriers that combine multiple properties in an allin-one system for drug delivery.114 They are used for the preparation of nanoparticles that have a gold core, an apolar polyester layer for drug loading, a polar PEO corona to provide biocompatibility, and three different types of surface reactive groups for surface functionalization (Fig 17). Uptake and subcellular localization of the nanoparticles can be controlled by multi-surface modification. The gold core ensures controlled degradation after cellular uptake115, the apolar polyester layer of the gold-stabilized particle provides a functional


loading space for apolar drugs, and PEO corona offers biocompatibility and biological stealthiness.116 It is also possible to control the surface density of targeting ligand, which in turn

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is important to achieve optimal selectivity.117 The cellular uptake with HeLa cells is reported by using different concentrations of the NPs (Fig 18). NPs loaded with oligo-arginine peptide show

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significant increase in cellular uptake in comparison to NPs with histidine-rich peptide. Former

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particles upload in the vesicular structures, whereas the latter find them in the cytoplasm and

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attach to the nuclear membrane. NPs loaded with anti-cancer drug exhibit dose-dependent

Fig 18. Cellular uptake and cytotoxicity of nanoparticles. (a).TEM images of representative sections of HeLa cells that were incubated with different kinds of NPs. The endosome is denoted as e and the cytosol as c. (b). Fluorescence images of cells incubated with Au NPs. (Reproduced with permission from ref 114)


cytotoxicity while the free anti-cancer drug is slightly more cytotoxic than the drug loaded

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nanoparticles because it takes a considerable period of time for the drug to escape the particles.

Gold nanorod-cored biodegradable block copolymer micelles for chemotherapy

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Several stimuli-sensitive biodegradable micelles have been developed to release drug in

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response to pH and temperature in tumor cells.118,119 Biodegradable micelles based on

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amphiphilic block copolymers of poly(ethylene glycol) (PEG) and aliphatic polyesters such as

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Fig 19. Illustration of gold nanorod-cored biodegradable micelles based on lipoylated poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL-LA) block copolymer for NIR-triggered release of doxorubicin (DOX) in cancer cells. (Reproduced with permission from ref 120) poly(ε-caprolactone) (PCL), polylactide (PLA), and poly-(lactide-co-glycolide) (PLGA) have received attention due to their biocompatibility in biomedical applications.120-122 It is due to their enhanced drug water solubility, improved drug bioavailability, decreased side effects, and increased tumor targeting ability via enhanced permeability and retention effect.123 Gold nanorod-cored biodegradable micelles prepared by coating gold nanorods (Au NRs) with lipoylated poly(ethylene glycol)-b-poly(ε-caprolactone) (PEGPCL-LA) block copolymer are used to release doxorubicin against cancer cells (Fig 19). It is interesting to note that these photoresponsive micellar formulations offer several unique advantages. They have uniform particle sizes and excellent colloidal stability under physiological conditions as PEG-PCLLA is covalently linked to Au NRs. They show good doxorubicin loading capacity and display slow drug release under physiological conditions efficiently into the nuclei of cancer cells, resulting in an effective reversal of drug resistance. The drug release on demand via near infrared (NIR) irradiation is carried out via photothermally induced phase transition of PCL regime from


ordered form to disordered form (Fig 20). NIR radiation, which is able to penetrate into the tissue up to 10 cm, has attracted a great interest for in vivo imaging and photothermal therapy, as well

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as remotely triggered drug release.124-128 In addition, NIR light allows precise temporal and spatial control over drug release rate and dosage. These gold nanorod-cored biodegradable

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micelles with low cytotoxicity, high stability, and remotely controlled drug release features are

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highly promising for targeted cancer chemotherapy.

Fig 20. Cell viability assays in MCF-7 cells. (A) NIR-enhanced antitumor activity of Au NR-MDOX. MCF-7 cells were incubated with Au NR-M-DOX, free DOX and Au NR-M for 4 h (DOX dosages: 1.0, 5.0, and 10 μg/mL), cultured in fresh media at 37 °C, irradiated by NIR laser (808 nm, 0.2 W/cm2) for 10 min, and then incubated for another 48 h before MTT assays; (B) Dependence of cell viabilities on DOX concentrations for MCF-7 cells treated with free DOX, Au NRM-DOX, and Au NR-M-DOX with NIR irradiation. (Reproduced with permission from ref 120) Block copolymer micelles – Au NPs for computed tomography (CT) guided radiation therapy


Au NPs loaded polymeric micelles can also be used for computed tomography (CT) guided radiation therapy treatment and radiosensitization.129 Au NPs have several advantages

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largely due to the high mass attenuation coefficient of gold, which is ∼2.7-fold higher than iodine129 normally used for such purposes. Even fine tuning of their size and shape of ∼30 nm

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can attenuate 120 kVp X-rays 5.7 times more than the iodine-based agents.130 Radiosensitization

Fig 21. In vivo CT images and intensity analysis of nu/nu nude mice with HT1080 flank tumors. (a) Representative CT images in the axial plane prior to injection (precontrast) and 30 min, 24 h, and 48 h post injection of Au NPs (n = 3) or AuroVist (n = 3). Tumor boundaries are indicated by white arrows. (b) Quantitative analysis of CT images. (Reproduced with permission from ref 129) works well with Au NPs due to their high absorbance which results in the deposition of energy from the photoelectric effect, Auger electrons, and the generation of free radicals.131,132 However, due to a rapid clearance of the small Au NPs, immediate irradiations of tumors are required after NPs administration. Thus, several critical factors need to be taken in care by the radiation oncologists while mapping of true tumor margins so that healthy cells are not affected, and hence mapping of the accurate definition of tumor boundaries should be precisely done before the delivery of radiation therapy.133-135 The radiosensitization provided by the Au NPs directly increases the radiation dose thus providing a second complementary mechanism by which the overall therapeutic index can be increased. Au NPs incorporated within the hydrophobic core of


multifunctional micelle that simultaneously exhibits long circulation times, achieves appreciable tumor accumulation, and generates CT image contrast, serves as a sensitizer for radiation therapy

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in cellular and animal models at sublethal radiation doses. A combination of CT-guided radiation therapy and gold-mediated radiosensitization improves the mean survival time of tumor-bearing

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mice in comparison to the one receiving radiation alone (Fig 21). A clear enhancement of the

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contrast with well-defined margins is observed at 24 and 48 h after the injection and provides excellent tool to differentiate between the tumor and normal tissue. This technique can further be improved to visualize regional tumor margins and spread, with minimizing exposure to normal

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tissue. Although, the contrast is not uniform throughout the tumor due to variations in the ability of Au NPs to penetrate beyond the vascular wall, nevertheless it helps clearly to demark the

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boundaries between the infected and normal tissues. Therefore, Au NPs based contrast agents are considered to be future wonder nanomaterials for guide and enhance the efficacy of radiation

Future perspectives

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therapy.

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Block copolymer micelles – Au NPs hybrid systems are expected to show enormous potential as drug delivery vehicles but there are inherent problems in the designing and characterizing such systems. Such systems are heterogeneous colloidal systems and hence, their emulsion stabilization in the aqueous phase is the most important aspect for precise target delivery systems. This is further associated with their polydisperse behavior for an appropriate induction in the systemic circulation as well as cell internalization. Finally, they should be prone to external stimuli such as temperature, pH, ionic strength, and irradiation in the event of drug release under specific circumstances that is obviously also related to their stability. Therefore, all these factors are needed to be taken in care while designing such systems weather they are based on in vitro synthesis or added effect of Au NPs to incorporate them in already block copolymer system in place. In contrast to much smaller micelles of conventional surfactants, the large shape and size of block copolymer micelles bear significant advantage in carrying large amount of drug for target delivery. While the former are practically incapable of carrying Au NPs because of comparable dimensions, block copolymer micelles on the contrary have the ability to carry a large number of tiny NPs as efficient contrast agents. Although, most of the water soluble block


copolymers are fairly non-toxic, familiarization with their nanotoxicity is another important factor in view of their appropriate usage in systemic circulation, oral delivery, or site specific

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injection.

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Concluding remarks

This account details the synthesis, characteristic features, and important biomedical

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applications of colloidal micelles of block copolymers – Au NPs hybrid morphologies as potential target drug delivery vehicles. The micelles of water soluble pluronics and tetronics

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demonstrate their ability as fine nanoreactors for the in vitro synthesis of Au NPs. The synthesis is mainly carried out by the surface cavities of these micelles and hence, compact, stable, and

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predominantly hydrophobic micelles prove to be more efficient nanoreactors than highly hydrated less hydrophobic micelles. As surface cavities produce Au NPs, they simultaneously

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self – assemble on the micelle surface due to colloidal stabilization provided by the block

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copolymer, thus a hybrid morphology is generated with unique characteristic features contributed by the soft micelle and metallic Au NPs.

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Soft micelle demonstrates its ability to respond to external stimuli such as temperature, pH, ionic strength, and irradiation effect, while Au NPs simultaneously act as contrast agents. A combination of both allows this hybrid morphology to precisely locate, target, and deliver the drug effectively with maximum and prolonged impact in minimum time with least side effects. This has been demonstrated by taking examples of various hybrid morphologies of different block copolymers with Au NPs and their important applications have been discussed in targeting cancer cells through cyctotoxicty by cell internalization, chemotherapy, and CT scan therapy.

Acknowledgment: These studies were supported by financial assistance under Article 27.9 of the CAS agreement of WLU, Waterloo.


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Colloidal Micelles of Block Copolymers as Nanoreactors, Templates for Gold

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Nanoparticles, and Vehicles for Biomedical Applications

100 nm

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Mandeep Singh Bakshi

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100 nm


Highlights Micelles (structured reducing agents) as nanoreactors

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Shape and size factors of nanoreactor micelles

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Multifuntional block polymer micelles – Au NPs

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Biodegradable block polymer micelles – Au NRs for chemotherapy

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Block polymer micelles – Au NPs for computed tomography (CT) guided radiation

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therapy

Liposomes as nanoreactors for the photochemical synthesis of gold nanoparticles.

Functionalized Gold Nanoparticles and Their Biomedical Applications.

Amphiphilic block copolymers-based mixed micelles for noninvasive drug delivery.

Magnetic nanoparticles as potential candidates for biomedical and biological applications.

Functional nanoparticles for biomedical applications.

Facile synthesis of gold and gold-based alloy nanowire networks using wormlike micelles as soft templates.

Synthesis of hollow mesoporous silica nanoparticles with tunable shell thickness and pore size using amphiphilic block copolymers as core templates.

Gold Nanoparticles: Recent Advances in the Biomedical Applications.

Magnetic nanoparticles adapted for specific biomedical applications.

Engineered magnetic nanoparticles for biomedical applications.

Biodegradable PEG-Based Amphiphilic Block Copolymers for Tissue Engineering Applications.

Towards the development of polycaprolactone based amphiphilic block copolymers: molecular design, self-assembly and biomedical applications.

Synthesis and controlled self-assembly of UV-responsive gold nanoparticles in block copolymer templates.

Rapid purification of gold nanorods for biomedical applications.

Convenient and effective ICGylation of magnetic nanoparticles for biomedical applications.

Radial growth of plasmon coupled gold nanowires on colloidal templates.

Toxicity and Biokinetics of Colloidal Gold Nanoparticles.

Block copolymer conjugated Au-coated Fe3O4 nanoparticles as vectors for enhancing colloidal stability and cellular uptake.

Highly stable, protein capped gold nanoparticles as effective drug delivery vehicles for amino-glycosidic antibiotics.

Tea-bag-like polymer nanoreactors filled with gold nanoparticles.

Gold-oxoborate nanocomposites and their biomedical applications.

Phytofabrication of bioinduced silver nanoparticles for biomedical applications.

Micelles of enzymatically synthesized PEG-poly(amine-co-ester) block copolymers as pH-responsive nanocarriers for docetaxel delivery.

DNA hydrogel as a template for synthesis of ultrasmall gold nanoparticles for catalytic applications.