Controlled design and construction of multifunctional nanoparticles by molecular self-assembly

Abstract

Controlled design of nanoparticles (NPs) displaying multiple functionalities is of great interest to many applications such as targeted drug or gene delivery, diagnostic imaging, cancer theranostics, delivery of protein therapeutics, sensing chemical and biomolecular analytes in complex environments, and design of future soldier protective clothing resembling a second skin. Current methods of synthesizing multifunctional nanoparticles (MNPs) typically involve sequential chemical processing of NPs; for example, drug-encapsulated NPs are first formed, followed by surface modifications involving the sequential conjugation of ligands to provide other functionalities such as targeting, responsiveness to stimuli, etc. We describe an alternate flexible approach to constructing MNPs employing the machinery of molecular self-assembly, starting with individually functionalized amphiphilic block copolymers. The commercially available polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer is used as the building block for illustrative purposes and functionalities are provided by other chemical moieties conjugated to it via degradable linkers. For demonstrative purposes, we have chosen folic acid (a targeting ligand), bovine serum albumin (resembling a therapeutic protein), and gadolinium (a MRI contrast agent) as the functionalities, but the choice of functionalities is not limited. The self-assembly of the conjugated block copolymers is induced by solvent polarity control, resulting in the production of MNPs. Quantitative determination of the amount of each conjugated functionality is done using spectrophotometry, which shows that the composition of the MNP is controlled by the composition of the precursor functionalized block copolymers and that self-assembly preserves the compositional control. The size of the MNP can be controlled by adding a second block copolymer. The combination of the ability to introduce multiple functionalities, vary the relative proportion of functionalities, and control the nanoparticle size, all independent of one another, renders the self-assembly approach uniquely efficient for producing interesting multifunctional nanoparticles for numerous applications.

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1. Introduction

The design of nanoparticles with multiple functionalities is one of the most active areas of research worldwide due to their potential applications as responsive sensors and as nanovehicles with targeting capabilities carrying drugs, DNA, imaging agents, etc., for theranostics applications.^1–7 The most common method for synthesizing multifunctional nanoparticles (MNPs) typically involves sequential chemical processing of nanoparticles, whereby the NPs are first prepared, followed by surface modifications through the serial conjugation of chemical moieties to provide desired functionalities. The conjugation of ligands requires the addition of excess amounts of reactants to ensure high conjugation efficiencies and consequently, the conjugated NPs need to be further purified by removing the excess reactants. Adding multiple functionalities onto a pre-existing NP is a challenging task because of the potential loss of one or more existing functionality (imparted in previous steps) due to the chemical processing steps involved in adding a new functionality. Currently, there are no established methods for the controlled introduction of multiple functionalities. Consequently, it is difficult to control the NP surface properties in a reproducible manner or to scale-up the production of such MNPs with well defined characteristics.

To precisely engineer MNPs in a simple way with scale-up capabilities, we suggest a bottoms-up approach using molecular self-assembly, starting from a collection of polymer molecules each of which is functionalized to impart a single functionality. In this paper, we demonstrate how the fundamental principles of self-assembly allow us to design, control and synthesize such MNPs. The multifunctionalities imparted to illustrate the concept include the ability to target tumor cells and serve as contrast agents for tumor diagnostics while exhibiting non-immunogenic response in the body. This is achieved by first preparing different individually functionalized block copolymers and then allowing their self-assembly to proceed to create multifunctional polymer NPs. The functionalized block copolymer was prepared by attaching the functional moiety to the end of a commercially available block copolymer via covalent conjugation. The approach allows us to control the surface functionalities and the composition of MNPs by controlling the composition of the functionalized block copolymers used as the starting building blocks. We also explore fundamental questions such as the stability of functionalized NPs after assembly and the role of different types of polymer in the assembly process.

In this study, we have developed MNPs whose surface contains targeting moieties that bind to receptors highly expressed in tumor cells while serving as contrast agents for tumor diagnostics. Commercially available amphiphilic polyethylene oxide–polypropylene oxide–polyethylene oxide triblock copolymers were used as the platform to construct the MNPs. Apart from their biocompatibility and ability to spontaneously form micelles in aqueous medium, these block copolymers have the advantage of being able to solubilize therapeutic agents. The versatility of their hydroxyl ending group enables easy chemical conjugation at that end, facilitating chemical modifications of the block copolymer.^8–10 Three chemical functionalities were added in this work: bovine serum albumin (BSA), folic acid (FA) and gadolinium (Gd). BSA, which is a blood protein, was used because it is stable, non-toxic, non-immunogenic, and exhibits a preferential uptake in tumor tissues.¹¹ Folic acid was chosen for its ability to selectively target tumor cells, which overexpress folate receptors.⁵ Gadolinium (Gd), which is highly paramagnetic with seven unpaired electrons and a long electronic relaxation time, was selected as a contrast agent. Free Gd³⁺ has been found to be toxic in both in vitro and in vivo studies. Although Gd³⁺ has been successfully complexed to organic chelators to overcome such problems, this complex has short half-life in blood and lack of specificity to target tumor tissues,¹² while the preparation of the chelators is tedious and relatively expensive. The key challenge in this research is not only to construct MNPs with controllable size and surface properties, but also to incorporate Gd³⁺ onto the NPs without the use of chelators. By taking advantage of self-assembly, NPs comprised of BSA and FA functionalized block copolymers have been successfully prepared with Gd³⁺ immobilized on the surface, through complexation with the COOH group on the functionalized Pluronic and amidazole group on BSA. The particle sizes and functionalities of the MNPs are conveniently controlled by manipulation of the ratio between each functionalized block copolymer used as the building block.

2. Experimental methods

2.1 Materials

A family of amphiphilic polyethylene oxide–polypropylene oxide–polyethylene oxide symmetric triblock copolymers with molecular weights in the range 2 kDa to 15 kDa and block composition in the range 20 to 80 weight percent ethylene oxide (available under the trade name Pluronics®) were obtained as a gift from the BASF Corporation. Two molecules from this family, F127 (MW 12 450 Da, 70% polyethylene oxide block and 64 propylene oxide units in the hydrophobic block) and P123 (MW 5750 Da, 30% polyethylene oxide block, and 70 propylene oxide units in the hydrophobic block) were used in our experiments. These two molecules have roughly similar lengths of the hydrophobic polypropylene oxide blocks. Other molecules from this family can be readily used to manipulate the size and shape properties of the nanoparticles synthesized since each block copolymer can give rise to a different size and/or shape depending on the molecular weight, block composition and polymer concentration. In this work, only size variations in the resulting nanoparticles are considered but not shape variations.

Succinic anhydride, 1,4-dioxane, dichloromethane, bovine serum albumin (BSA), N,N′-carbonyl diimidazole (CDI), folic acid (FA), 1,2-ethylenediamine, triethylamine (TEA), acetone, hexane, acetonitrile, diethyl ether, dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Company. 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) was purchased from ACROS Chemical Company. N-Hydroxy succinimide (NHS) and 4-dimethyl aminopyridine (DMAP) were purchased from Alfa Aesar. Nanopure water and phosphate saline buffer (PBS 1×, pH 7.4) were used throughout the experiment, unless specified otherwise.

2.2 Synthesis of COOH-terminated F127 (F127-COOH) and P123 (P123-COOH)

The synthesis was conducted following previous reports.¹³ Briefly, F127 (6.3 g; 1 mmol OH) and DMAP (122.17 mg; 1 mmol) were dissolved in 1,4-dioxane (15 mL) in the presence of TEA (139 μL) and stirred under nitrogen for 30 min. Succinic anhydride solution (125 mg in 5 mL 1,4-dioxane) was then added dropwise to the copolymer solution while stirring. The solution mixture was left stirring at room temperature for 24 h. The excess 1,4-dioxane was removed by rotary evaporation, and the remaining samples were precipitated three times in cold diethyl ether while stirring. The precipitate was dried under vacuum overnight at room temperature to give the white powder of F127-COOH. The presence of COOH was analyzed using FT-IR and ¹H-NMR. The procedure for P123-COOH was identical with P123 as the starting point instead of F127.

2.3 Synthesis of NHS-terminated F127 (F127-NHS)

In this step, the carboxyl terminated F127 was activated with NHS. Briefly, F127-COOH (500 mg; 0.08 mmol) and EDC (77 mg; 0.4 mmol) were dissolved in 5 mL dichloromethane in a round-bottom flask. After stirring the mixture for 30 min, NHS (46 mg, 0.4 mmol) was added, and the solution mixture was left stirring at room temperature for 24 h. Next day, the solution mixture was precipitated three times in cold diethyl ether. The precipitate was further dried under vacuum overnight at room temperature to give the white powder of F127-NHS. The presence of NHS was confirmed using FT-IR and ¹H-NMR.

2.4 Synthesis of BSA-conjugated F127 (F127-BSA)

The strategy for attaching BSA to F127 was adapted from previous literature.¹⁴ BSA in PBS solution (30 mg mL⁻¹) was added dropwise into the F127-NHS solution (15 mg mL⁻¹) while stirring. Then, the reaction mixture was allowed to stir at 4 °C for 48 h. The samples were centrifuged at 8000 × g to 16 000 × g for 20 min to remove excess BSA, followed by resuspension in water. The sample was lyophilized and stored at 4 °C until use. The purification process was monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE® Novex 12% Bis-Tris Gel (Molecular Probes, Invitrogen) with MOPS (3(N-morpholino)propanesulfonic acid) running buffer (Invitrogen). The gel was stained with Coomassie Blue prior to visualization. When no more free BSA was detected, the reaction mixture was lyophilized and kept at −20 °C.

2.5 Synthesis of CDI-terminated F127 (F127-CDI)

F127-CDI was prepared following a procedure described previously.¹⁴ F127 was first purified by dissolving in acetone, precipitating in cold hexane, and drying under vacuum at room temperature. The purified F127 (5.0 g; 0.4 mmol) was then dissolved in dry acetonitrile (7 mL) and added dropwise to a solution of N,N′-carbonyl diimidazole (CDI) (0.65 g; 4 mmol) in dry acetonitrile (7 mL) at room temperature under nitrogen. The reaction mixture was allowed to stir overnight and concentrated using a rotary evaporator. The concentrated mixture was purified by precipitating in excess of cold diethyl ether while stirring. This process was repeated at least three times to ensure that the excess CDI was completely removed. The purified F127-CDI was dried under vacuum overnight at room temperature and collected as white powder.

2.6 Synthesis of amine-terminated F127 (F127-NH₂)

A solution of F127-CDI (2.1 g; 0.16 mmol) in acetonitrile (2.5 mL) was added dropwise to 1,2-ethylenediamine (2 mL) at room temperature over 2 h. The reaction mixture was allowed to stir overnight, and the excess 1,2-ethylenediamine was removed by rotary evaporation. The crude product was further purified by precipitating in excess cold diethyl ether. This process was repeated three times. The purified F127-NH₂ was dried under vacuum overnight at room temperature and collected as white powder.

2.7 Synthesis of folate-conjugated F127 (F127-FA)

The synthesis of F127-FA was conducted following the procedure as previously reported.¹⁴ Briefly, under nitrogen atmosphere, triethylamine (50 μL) and EDC (10 mg; 0.0522 mmol) were added to a DMSO solution (1 mL) containing folic acid (FA) (11 mg; 0.0237 mmol). After 1 h stirring in dark, NHS (6 mg; 0.0522 mmol) was added and allowed to stir for 1 h, followed by the addition of F127-NH₂ (100 mg; 0.0079 mmol). The reaction mixture was allowed to stir overnight, in dark, at room temperature, diluted in water and dialyzed against water for 3 days using Spectra/Por 3 Dialysis Membrane (MW cutoff 3500 Da). The purified sample was subjected to lyophilization to obtain yellow powder.

2.8 Preparation of MNPs

MNPs were prepared by two alternate procedures, using conventional stirring. In one method of preparation (Method I), the lyophilized or solid samples of the functionalized or unfunctionalized block copolymers (F127-BSA, F127-FA, P123-COOH, and P123) were all combined in appropriate amounts as solids and then dissolved together in nanopure water to create the MNP. In the alternate method (Method II), each solid sample was first dissolved separately in water to create a solution of one type of micelles and the different micellar solutions were then mixed to create the MNP.

In a typical procedure, P123, F127-BSA, and F127-FA were mixed in water at various mass ratios to the final concentration of 1% (w/v). The solution mixture was allowed to stir at room temperature for about 1–2 h under dark. The sample was filtered through 0.45 μm filters (Millipore) prior to the DLS analysis, and the sample was centrifuged at 5000 × g for 5 min to concentrate the samples prior to the UV-Vis analysis.

For the synthesis of Gd-MNP, the prepared mixed micelle solution of P123-COOH, F127-BSA, and F127-FA was added dropwise to the solution of GdCl₃·6H₂O (10 mg mL⁻¹). The mixture was gently stirred overnight at 4 °C under dark. The Gd-MNP micelles were purified by centrifugation at 5000 × g for 5 min, and the pellet was immediately resuspended in ice cold water. The step was repeated at least 3 times to ensure that the free Gd was removed. The presence of Gd was confirmed by EDAX (Energy-dispersive X-ray spectroscopy), and the mass percent content of Gd(III) on MNP was quantified using ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry).

2.9 Stability measurements

For the study of the salt effect on the stability of the MNPs (as determined by the aggregation tendency of the MNPs), the experiments were conducted as described earlier but with 0 M to 1 M NaCl or 1.0 M CaCl₂ as the solvent instead of water.

For studying the effect of lyophilization on MNP stability, the first cycle of experiments was performed by dissolving in water the lyophilized sample that had been stored for two months. The solution from cycle 1 was again lyophilized and the solids were stored for one month and then after redissolution of the solids in water, the second cycle of experiments was performed. The solution from cycle 2 was then lyophilized again and the solids were stored for two weeks and then were redissolved in water for conducting the third cycle of experiments.

2.10 Characterization methods

¹H-NMR was acquired on a Bruker ARX 500 MHz spectrometer. Infrared spectroscopy was recorded on a Perkin-Elmer Spectrum One Fourier transform IR (FTIR). The hydrodynamic diameter and size distributions were measured with dynamic light scattering (DLS) using the Zetasizer Nano ZS (Malvern Instruments). Scanning electron microscopy (SEM) was performed on the Zeiss EVO-60 SEM. Molecular weight of F127-BSA was determined using MALDI-TOF mass spectrometer. Pure BSA was used as a reference sample. The surface charge densities (ζ-potential values) and scattering intensity were measured using a Zetasizer Nano ZS (Malvern) at 25 °C. The scattering intensity was measured at a scattering angle of 173° relative to the source. The zeta potential was measured using a dip cell. All samples were diluted 100 times in water prior to the ζ-potential measurements to avoid any interference from counter ions.

TEM images were obtained on JEOL 2010 TEM equipped with a LaB6 (lanthanum hexaboride) filament, operated at 200 keV with GIF 2001 spectrometer and 1 megapixel CCD camera. For sample preparation, 10 μL of each sample was dropped on a copper grid and allowed to evaporate overnight. Then, 10 μL of phosphotungstic acid (PTA) solution (2% w/v) was added to the grid. After 2 min, the solution was drawn off from the edge of the grid with filter paper, and the grid was allowed to air dry prior to the analysis. The particles containing Gd³⁺ were visualized without further staining.

3. Results and discussion

3.1 Synthesis of BSA-conjugated F127

Synthesis steps. The synthesis of BSA-conjugated F127 consisted of three major steps as shown in Fig. 1A. The Pluronic block copolymer is chemically inert and not ready to be conjugated to proteins. Therefore, first, F127 was activated with succinic anhydride to form carboxylated-conjugated F127 (F127-COOH). Second, N-hydroxy succinimide (NHS) was introduced to the F127-COOH to create a good leaving group for enzyme conjugation (F127-NHS). Third, the amino group of lysine on the BSA reacts with F127-NHS via addition/elimination to create stable amide bond between F127 and the BSA (F127-BSA).

Fig. 1 (A) Schematic illustration of the synthetic steps in the preparation of bovine serum albumin (BSA)-functionalized F127 (F127-BSA). (B) FT-IR and (C) H-NMR spectra of F127, F127-NHS, and F127-COOH, respectively. The presence of COOH and NHS are confirmed by FT-IR and H-NMR as explained in the text.

Confirming end functionalization of F127. The successful preparation of F127-COOH and F127-NHS were confirmed using FT-IR and ¹H-NMR (Fig. 1B and C). The NMR spectrum of F127-COOH shows a characteristic peak at ∼2.6 ppm, corresponding to the protons on the CH₂–CH₂ of the succinic anhydride. FT-IR spectrum shows a prominent peak at ∼1730 cm⁻¹, corresponding to the stretching of the carbonyl group on COOH. For the F127-NHS, the protons on the CH₂–CH₂ of the succinic anhydride were deshielded to ∼3.2 ppm while a prominent peak at ∼2.8 ppm corresponds to the CH₂–CH₂ of the NHS ring. The FT-IR spectrum shows a characteristic peak of the carbonyl and amide bonds at ∼1713 cm⁻¹, and 1570 cm⁻¹, respectively. The results from both NMR and FT-IR confirm a successful activation of F127. The results confirm that end-functionalized block copolymers can be successfully prepared from simple chemical reactions.

Confirming conjugation of BSA to F127. A successful conjugation of BSA to F127 was confirmed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Fig. 2A shows SDS-PAGE profiles of the following samples: (i) Mark 12 standard protein, (ii) standard BSA, (iii) and (iv) F127-BSA after extensive purification steps. The major band at 66.5 kDa is associated with the BSA while the bands at a higher molecular weight are associated with F127-BSA (F127 is approximately 12.5 kDa). The excess BSA was effectively removed by centrifugation.

Fig. 2 (A) SDS-Page analysis, Lane 1-Standard protein Mark 12, Lane 2-Native unmodified BSA (∼66 kDa), Lane 3 and 4-F127-BSA after extensive purifications. (B) MALDI-TOF mass spectrum of native unmodified BSA (Top) and F127-BSA (Bottom). The results reveal a successful conjugation of about 1 : 1 F127:BSA.

The molecular weight of F127-BSA was also confirmed by MALDI-TOF mass spectrometry (Fig. 2B). The MALDI was conducted in 70% acetonitrile where the micelles will undergo disassembly to singly dispersed BSA-conjugated F127 block copolymer. In MALDI-TOF, the primary species correspond to singly ionized state of the protein while a small amount of doubly ionized specie is also generally obtained. The peaks at 66.4 and 33.3 kDa obtained for BSA represent the unmodified BSA in the singly and doubly ionized states, respectively. The peak at 79.6 kDa obtained for F127-BSA suggests that F127 was conjugated to BSA with approximately 1 : 1 ratio, since that will correspond to the 1 : 1 conjugate in its singly ionized state. The peak at 39.9 kDa obtained for F127-BSA again corresponds to the 1 : 1 conjugate, but in the doubly ionized state. No peak for unmodified BSA was seen for F127-BSA, indicating the efficiency of purification.

The presence of protein as a conjugate affects its UV-Vis spectrum in comparison to that of the free BSA in solution. A blue-shift of about 3 nm was observed after the conjugation indicating a slight change of the microenvironment polarity around the tryptophan and/or tyrosine residues of BSA (Fig. 3).¹⁵

Fig. 3 UV-Vis spectrum of multifunctional NPs at different mass ratios of P123/F127-BSA/F127-FA. A blue-shift of about 3 nm was observed after the conjugation indicating a slight change of the microenvironment polarity around the tryptophan and/or tyrosine residues of BSA.

Assessing the degree of polymer conjugation. Because BSA has multiple lysine sites on the surface for conjugation, a number of conjugates with the end-functionalized F127 are potentially possible. Some conjugates and their anticipated approximate molecular weights for singly and doubly ionized states are as follows: (i) only one end of F127 is conjugated to a molecule of BSA (1 : 1 conjugate, 79 and 39.5 kDa), (ii) each end of F127 is conjugated to a different BSA molecule (1 : 2 conjugate, 145 and 72.5 kDa), (iii) both ends of F127 are conjugated to the same BSA molecule (1 : 1 conjugate, 79 and 39.5 kDa), (iv) two F127 molecules are conjugated to the same BSA molecule (2 : 1 conjugate, 92 and 46 kDa), and more complex conjugates involving multiple BSA and multiple F127 molecules. The type of conjugates formed depends upon the proximity of the lysine residues on BSA, the size of the block copolymer molecule as well as the concentrations of the protein and the block copolymer in solution. A theory for the degree of conjugation to explain the observed 1 : 1 conjugation remains to be developed. The SDS-PAGE and MALDI-TOF cannot resolve whether only one end or both ends of F127 are covalently bound to one BSA molecule since both options give rise to the same mass peaks.

3.2 Synthesis of folate-conjugated F127

The synthesis of F127-FA consisted of three major steps (Fig. 4A). ¹H NMR spectrum (300 MHz, CDCl3, ppm) of F127-CDI was similar to previously reported (data not shown). The peaks at 7.4, 7.7, and 8.9 ppm correspond to the protons on the imidazole rings. As shown in Fig. 4B, FTIR spectrum confirmed the presence of carbonyl groups in the polymer chain (1713 cm). ¹H NMR spectrum of F127-NH₂ illustrated the characteristic peaks at ∼2.8 and 3.2 ppm, corresponding to the protons on the ethylamine. ¹H NMR spectrum of F127-FA shows characteristic peaks of FA between 6–9 ppm, indicating a successful conjugation (Fig. 4C). The amount of FA conjugated onto F127 was determined by constructing a calibration curve of folic acid in DMSO using UV-Vis. The conjugation percentage of FA to F127 was shown to be about 72% on a molar ratio basis.

Fig. 4 (A) Schematic of the synthetic steps for the preparation of folic acid (FA)-functionalized F127 (F127-FA). (B) FT-IR and (C) H-NMR spectrum of F127, F127-NH₂, F127-CDI, and PF127-FA, respectively.

3.3 Self-assembly of MNPs

The synthesis of multifunctional NPs was conducted following two different approaches. In one approach, the lyophilized samples of the two functionalized block copolymers (F127-BSA and F127-FA) and the solid unfunctionalized block copolymer P123 were all mixed first as solids and then dissolved together in nanopure water allowing them to spontaneously self-assemble into spherical MNPs (Fig. 5A). Both F127 and P123 have comparable lengths of the hydrophobic block, allowing good mixing in the micelle core and permitting manipulation of the micelle size through variations in the relative amounts of the two block copolymers. Upon mixing in aqueous solution, the micelles were formed through hydrophobic interaction of the PPO units of the functionalized F127 and P123, resulting in relatively narrowly dispersed micelles with various functionalities on the surface (Fig. 5B).

Fig. 5 (A) Schematic representation of the formation of functionalized block copolymer micelles comprised of P123, folic acid-functionalized F127 (F127-FA), and bovine serum albumin-functionalized F127 (F127-BSA). These MNPs can be prepared via either (i) the direct assembly of mixtures functionalized block copolymers (Method I) or (ii) by mixing the preformed micelles generated from each block copolymer (Method II). TEM images of F127-MNP1 (B), F127-BSA (C), and F127-FA (D). For TEM images, scale bar = 200 nm. Size distribution from DLS for P123, F127, F127-FA, F127-BSA micelles (E) and for mixed micelles of F127-FA + F127-BSA (Method II), and MNP1 prepared by direct assembly (Method I) or by mixing pre-formed micelles (Method II) (F).

In the second approach, the lyophilized samples of the functionalized block copolymers (F127-BSA and F127-FA) were each separately dissolved in the nanopure water and allowed to self-assemble into micelles (Fig. 5C and D). The MNPs were then prepared by mixing these two kinds of micelles in solution with a third solution of micelles formed of the unfunctionalized block copolymer P123. The nanoparticle formation occurs as a result of spontaneous self-assembly. Since the nanoparticles generated are equilibrium structures, they undergo size changes as a function of the composition. As a result, the mixing of micelles of different sizes can lead to nanoparticles of a size different from those of starting micelles.

3.4 Size control of MNPs

The presence of MNPs and micelles was qualitatively observed from TEM imaging (Fig. 5B–D) and their sizes could be roughly estimated to be approximately 30 nm for MNP1, 100 nm for the F127-BSA micelles and about 150 nm for the GF127-FA micelles. The particle sizes were quantitatively determined using dynamic light scattering (Fig. 5E and F). The DLS results for non-functionalized F127 show one peak at about 4 nm for the non-micellar, singly dispersed molecule of F127 and a second peak at about 60 nm for F127 micelles (Fig. 5E). The DLS data show that P123 micelles have a diameter of about 20 nm.

The fact that both F127-BSA and F127-FA can separately self-assemble into micelles is confirmed by the DLS data in Fig. 5E. The F127-BSA micelles have a size of about 90 nm (Fig. 5E), and the micelle size is larger than that of the unmodified F127 micelles by about 30 nm. The shape of BSA in solution has been discussed in the literature in the context of interpreting scattering data for BSA, taking into consideration the X-ray crystallographic data available for human serum albumin.^16–18 The shape has been suggested to be a prolate ellipsoid with dimensions of 14 nm × 4 nm for the major axes, with three domains aligned along the long axis. The X-ray crystallographic data have indicated a heart-shaped structure. Recently, an equilateral, triangular prismatic shell shape with a side length of 8.4 nm and a thickness of 3.2 nm has been proposed. If either the prismatic shell or the prolate ellipsoidal shapes are considered, then the presence of the BSA at the micelle surface would account for a size increase of about 17 to 28 nm. The further small increase in the diameter for the F127-BSA micelles could imply that the conjugation to BSA has caused an intrinsic change in the micelle aggregation number compared to the free F127 micelles.

In Fig. 5E, the size distribution of F127 micelle shows some tailing towards bigger size regime (>1000 nm) and also shows increasing intensity in the microns size range. To understand this distribution, we note that the direct result from a DLS experiment is an intensity distribution of particle sizes. The intensity distribution is weighted according to the scattering intensity of each particle fraction. For example, if we have a mixture of equal numbers of 10 and 100 nm size particles, then both will have the same peak areas in the number distribution; the 100 nm particle will have 10³ times larger peak area in the volume distribution; and the 100 nm particle will have a 10⁶ time larger peak area in the intensity distribution. Thus the presence of a dust particle or agglomerate can appear as significant peak of large area in the intensity distribution, typically if the particle size appears to be 1 micron or above. No quantitative information is usually extracted from light scattering data for sizes of about 1 micron or larger, other than recognizing the presence of some dust or agglomerates. The mass of polymer in such large agglomerate may still be negligible.

The DLS data for F127-FA micelles indicate a broader size distribution and larger average micelles size at about 200 nm compared to the F127 micelles (Fig. 5E). Clearly, this should imply that folic acid contributes to interactions that favor the micelle growth. Folic acid is composed of three primary structures: 2-amino-4-hydroxy-6-methyl pteridin, para-aminobenzoic acid, and L-glutamic acid. Since it is comprised of hydrophilic carboxyl and amino groups, it can interact via hydrogen bonding.¹⁹ Therefore, we speculate that attractive interactions between folic acid moieties in the micelle may lead to micellar growth and a broader size distribution.

The F127-BSA molecules and the F127-FA molecules were both mixed in the ratio 2.5 : 0.5 along with P123 to create the multifunctional nanoparticle, MN1 by both Methods I and II. The size distributions of MNP1 from both methods are shown in Fig. 5F. Also shown is the size distribution for the binary mixture of F127-BSA + F127-FA in the same ratio 2.5 : 0.5, prepared by Method II. This binary mixture differs from MNP1 only in the absence of P123. The size distribution of F127-BSA and of F127-FA both show single populations while for the F127-BSA + F127-FA mixture there are two populations (ignoring any peaks in the micron size range as mentioned in previous comment). The first population of the mixture has a size smaller than those of F127-BSA micelles and F127-FA micelles, while the second population of the mixture is somewhat larger than the size of F127-FA micelles. The type of interactions possible with folic acid as well as the known reduction in the electrostatic interactions between folic acid and BSA may contribute to the large size and broader size distribution.

Also shown on Fig. 5F are the sizes of the MNP formed from a ternary mixture of F127-BSA, F127-FA and P123, based on the two approaches for MNP formation described earlier. One involves combining all three copolymer solids and mixing them together in water to directly generate the MNPs. The other is to mix each of the solid in water to create three micellar solutions first and then mix the three micellar solutions to generate the MNPs. Both methods provided practically the same size for MNPs. Both systems have the same composition 6/2.5/0.5 for the components P123/F127-BSA/F127-FA. One may note that the mixed micelles generated for the composition 2.5/0.5 of the binary system F127-BSA/F127-FA gave rise to very large micelles as can be seen from Fig. 5F. When these two kinds of micellar solutions were mixed with a solution of P123 micelles, the resulting MNPs were very narrowly dispersed in their sizes, closer to the size of the P123 micelles. The presence of P123 in large enough concentrations in the micelle would reduce the chances for hydrogen bonding involving F127-FA molecules, thereby decreasing the attractive interactions that were responsible for the large aggregates to form.

3.5 Size and composition control of MNPs

The self-assembly process has been shown to provide MNP size control in Fig. 5F. To explore this further, we determined the size of MNPs for binary and ternary mixtures of copolymers.

Fig. 6A shows the size distributions for the micellar NPs consisting of different mass ratios of P123 and F127-BSA. The P123 micelles have the smallest particle sizes (about 20 nm) whereas the F127-BSA micelles have larger and more broadly dispersed sizes at about 90 nm. The DLS data show that the average size of the NPs decrease as the content of P123 increases, and also there is a corresponding decrease in the polydispersity. While this MNP size control through addition of copolymer P123 is shown for illustrative purposes, we note that a wide range of copolymers from the same family are available with ability to form aggregates of different sizes and shapes.²⁰ They can be readily mixed because of their mutual compatibility with one another, thereby providing a convenient handle to tune MNP sizes and shapes.

Fig. 6 (A) Size distribution of P123 : F127-BSA mixed micelles at different mass ratios starting from pure P123 micelles (1 : 0) to pure F127-BSA micelles (0 : 1). Both the average size and the polydispersity index (PDI representing the width of the size distribution) increased as the F127-BSA content increased. (B) The percent content of BSA and FA on the final particles after centrifugations and resuspensions. High contents of BSA (>75%) and FA (>80%) still remained on the NPs even after assembly and regardless of the NP size. This figure corresponds to Table 1.

We evaluated the compositional control over the size of MNPs comprised of FA and BSA by preparing the MNPs using various mass ratios of P123/F127-BSA/F127-FA as indicated in Table 1.

Table 1 Composition of Multifunctional Nanoparticles (MNP)^a

Sample	P123/F127-BSA/F127-FA	P123/F127
a P123/F127-BSA/F127-FA denotes the mass ratio between the three functionalized block copolymers involved. The ratio P123/F127 denotes the mass ratio between the copolymers independent of functionalization.
MNP1	6.0/2.5/0.5	6.0/3.0
MNP2	4.5/3.5/1.0	4.5/4.5
MNP3	3.0/5.0/1.2	3.0/6.2

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Comparison of the sizes of P123/F127-BSA MNPs in Fig. 6A against that of P123/F127-BSA/F127-FA in Fig. 6B shows that the sizes are primarily determined by the overall ratio of P123/F127 rather than by the functionalities.

The composition control over the MNPs were confirmed by using spectrophotometry to determine the content of BSA and FA on the final MNP structures in comparison to the initial amount used before assembly. Fig. 6B shows that over 75% of the expected BSA and over 80% of the expected FA remained on the MNPs regardless of the NP size or composition (theoretically 100% of the original amounts of BSA and FA should be recoverable). From these results, we can conclude that the challenge associated with controlling the amount of each component in the final MNPs is governed by ratio of the starting copolymers and that self-assembly is a suitable technique for controlling the surface composition of the MNPs.

3.6 Stability of MNPs

Solution stability. A major concern with the use of NPs for most applications is the potential for nanoparticle aggregation after a period of time. Hence, the stability of the MNPs was investigated by monitoring the changes in MNP size over time, selecting MNP1 as a model system. The results of storage of the MNP solution for 2 months at 4 °C demonstrated that although some aggregation was observed after 30 days, there were statistically insignificant changes in the size of MNPs (Fig. 7A).

Fig. 7 (A) The size distribution of MNP1 after storing at 4 °C for different time periods. The aggregation of NPs was observed after 30 days of storage. (B) The size distribution of MNP1 after lyophilization and redispersion in water. No significant change in size of the NPs was observed after the first two cycles of lyophilization. Some aggregation was observed in the third cycle likely due to the denaturation of the BSA.

Long term storage stability. In order to maintain the stability over long shelf-life of the MNPs, lyophilization was explored. For studying the effect of lyophilization on MNP stability, the first cycle of experiments was performed by dissolving in water the lyophilized sample that had been stored for two months. The solution from cycle 1 was again lyophilized and the solids were stored for one month and then after redissolution of the solids in water, the second cycle of experiments was performed. The solution from cycle 2 was then lyophilized again and the solids were stored for two weeks and then were redissolved in water for conducting the third cycle of experiments. Fig. 7B shows no significant change in size of the MNPs after the first two cycle of lyophilization. Small aggregations were observed in the third cycle, probably due to the denaturation of the BSA.

Stability against ions. Further, the effect of ions on the stability of the MNPs was investigated. The MNP self-assembly was conducted at various concentrations of NaCl. At salt concentrations of about 0.2 M NaCl, aggregation of the MNPs become observable (Fig. 8) and the propensity for aggregation increases with increasing concentrations of NaCl. We anticipate that the presence of the counter ions screens the surface charge of the functionalized MNPs, reducing the stabilizing repulsive force between the MNPs, which ultimately leads to aggregation. Similar significant aggregation behavior was observed when strongly hydrated divalent cation salt, CaCl₂ was used.

Fig. 8 Salt effects on the stability of MNP1. The aggregation increases as the salt concentration increases. It is anticipated that the cationic salt may screen the surface charge of the particles, preventing the stabilizing repulsive force between each MNP which ultimately leads to the aggregation. The measurements were performed in triplicate, and the average and standard deviation were reported.

3.7 Synthesis and characterization of Gd-NP complexes

To synthesize the Gd-NP complexes without the use of low molecular weight Gd³⁺ ligands, NPs with enough chelating groups on the surface must first be prepared. Since previous studies²¹ have shown that Gd has an affinity for both carboxyl and amidazole groups on serum albumin, the NPs were designed to have both of these on the surface. The synthesis of such NPs was performed in a similar manner as described previously, but with P123 replaced by P123-COOH. The solution was maintained at pH 7.4 to prevent GdCl₃ from being hydrolyzed.¹² The Gd³⁺ was introduced after the NPs were formed. Upon overnight incubation followed by centrifugations to remove excess Gd³⁺, the Gd³⁺ was successfully bound to the surface of the NPs.

The TEM analysis showed a uniform size distribution of spherical structures with the size of ∼250 nm in diameter, consistent with the results obtained from DLS (Fig. 9B). The size of the Gd-NP complex was greater than that of the unbound NPs, and the size increases as the concentration of Gd³⁺ (0–0.05 M) increases to the point where precipitation was evident (data not shown). It is anticipated that the size increase upon the presence of Gd might be due to the same effect as when adding NaCl or CaCl₂. Higher valency cations are more effective in neutralizing the net charge and weakening the repulsive interactions between particles. Hence, precipitation was observed when using a low concentration of GdCl₃ (0.05 M). Different control studies were conducted to further understand the Gd³⁺ binding mechanism. Both EDAX and ICP-OES showed no Gd³⁺ binding when unmodified Pluronic micelles were used (Fig. 10). However, when COOH was introduced to the micelles, the Gd³⁺ binding ability increased to about 10–16%. A similar result was observed when F127-BSA was used. The results suggested the mechanism of Gd binding to NPs is mainly based on the coordinate covalent linkage with COOH on the functionalized Pluronic and amidazole on BSA (Table 2).

Fig. 9 (A) Schematic illustration of the formation of gadolinium (Gd)-modified MNP1. The Gd was introduced after the nanoparticle was formed. (B) TEM images of GD-MNP1 prepared with P123, (C) TEM images of GD-MNP1 prepared with P123-COOH, (D) size distribution of GD-MNP1 prepared with P123, by DLS, (E) size distribution of GD-MNP1 prepared with P123-COOH, by DLS. The size of both Gd-MNP1complexes were significantly greater than that of the MNP1 indicating that the charge neutralization due to Gd binding to the surface of the MNP1 allowed for reversible self-assembly to generate nanoparticle of a very different size compared to the MNP1 lacking bound Gd. For the TEM images, scale bar = 500 nm.

Fig. 10 EDAX analyses of Gd on different types of NPs. The results show that a relatively high content of Gd was evident when MNP1 (P123/F127-BSA/F127-FA) and COOH-functionalized NP (P123-COOH/F127-COOH) were used. A small amount of Gd was observed when F127-BSA micelles were used. In contrast, no Gd was observed when non-functionalized Pluronic micelles were employed. The results suggest the mechanism of Gd binding to NPs is mainly based on the coordinate covalent linkage with COOH on the functionalized Pluronic and amidazole on BSA.

Table 2 ICP-OES analyses of the Gd content on MNPs and different control studies

Sample	% GD ON NP
P123-COOH/F127-COOH	16.8963
P123/F127	−0.8035
MNP1 with P123-COOH	31.271
F127-BSA	16.2576
BSA	6.3237

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The presence of Gd³⁺ was further confirmed by measuring the surface charge densities of the complex (Fig. 11). At pH 7.4 both P123 and F127 were slightly negatively charged with zeta potentials of −6.8 and −8.7 mV, respectively, which is common for neutral polymeric NPs.²² Anionic micelles, such as P123-COOH or F127-COOH exhibit negative charge, yielding an overall negative zeta potential on the NP surface. Following treatment with Gd, the zeta potential becomes positive, confirming the presence of Gd on the surface of the NPs.

Fig. 11 Zeta-potential values of MNP1, Gd-MNP1, and other control studies in PBS solution at PH 7.4. The most negative charge was observed when COOH was functionalized on the surface of the NPs. Upon the addition of Gd, the zeta potential increased to the positive values, confirming the presence of Gd on the surface of the NPs.

4. Conclusions

In this study, molecular self-assembly has been used to create a versatile platform for constructing multifunctional NPs comprised of non-immunogenic proteins, targeting motif, and a contrast agent. The process starts with the conjugation of different moieties (FA and BSA) onto the amphiphilic block copolymer, allowing self assembly to take place, along with the ligand-type interaction to allow Gd to be present on the final NP.

A single Pluronic such as F127 is sufficient to generate the multifunctional nanoparticle and any of the Pluronic block copolymers available commercially (some 20 or so molecules) can be activated and functionalized to generate the MNPs by following the methods developed in this manuscript. We have the added advantage that different Pluronic molecules readily mix with one another. Since each Pluronic molecule forms an aggregate of different size and/or shape, one can manipulate the size and shape of nanoparticles by combining different Pluronics.

In this work we have used P123 as an additive to change only the size of the nanoparticle, but the use of P123 is not unique. Since each Pluronic molecule forms an aggregate of different size and/or shape, one can manipulate the size and shape of nanoparticles by selecting suitable Pluronics. The block composition (relative size of the PEO and PPO blocks) and polymer concentration control the formation of cylindrical and lamellar structures and phase equilibrium data for Pluronics showing such aggregate morphologies are available in the literature.²⁰ Although we have not chosen the Pluronic molecule to achieve shape changes in this study, changes to cylindrical and lamellar particles are achievable with suitable choices of Pluronics.

The surface composition of the NP can be controlled by altering the mass ratio of each functionalized block copolymer and the amount of Gd added. It is easy to increase the loading of the therapeutic protein just by increasing the relative proportion of the protein conjugated block copolymer.

The synthesis approach offers the unique possibilities of controlling the size, composition, and shape (though not demonstrated in this study) of the multifunctional nanoparticles, all independent of one another through simple molecular choices.

The functionalized NPs were shown to be stable when stored in aqueous medium for about one month while the shelf life was increased to four months or longer when stored as a dry formulation. This work shows the feasibility of assembling different functionalized block copolymers to generate multifunctional NPs that can be tailor designed in terms of their size and functionalities for applications in drug delivery, diagnostic imaging or chemical and biological molecular sensing.

Acknowledgements

This work was supported by the Defense Threat Reduction Agency (DTRA) Project #BA12PHM159 and the NSRDEC In-House Laboratory Independent Research (ILIR) program. Nisaraporn Suthiwangcharoen is the recipient of the National Research Council Chemical and Biological Defense (NRC/DTRA CBD) Research Associateship. We would like to thank Prof. Qian Wang and Jittima Luckanagul at the University of South Carolina for use of the ICP-OES.

Notes and references

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