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Apr 13, 2017 - Interfaces , 2017, 9 (17), pp 14647–14655 ... I-radiolabeled NaYF 4 :Yb 3+ /Er 3+ @PEG nanoparticles for multimodal in vivo tissue im...
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Polymer-Coated Ultrastable and Biofunctionalizable Lanthanide Nanoparticles Yurong Que, Chun Feng,* Guolin Lu, and Xiaoyu Huang* Key Laboratory of Synthetic and Self-Assembly Chemistry for Organic Functional Molecules, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, People’s Republic of China S Supporting Information *

ABSTRACT: Lanthanide-containing nanoparticles (LnNPs), for example, NaYF4, are considered to be one of the most promising platforms for biological and material applications due to their unique and unusual magnetic and upconversion fluorescent properties. However, limited water dispersity, low long-term colloidal stability, and difficulty in further functionalization greatly narrow the scope of their application in the real world. Herein, we report a facile strategy to counter the aforementioned barriers to the expanding use of LnNPs that involves surface-coating the LnNPs with poly(ethylene glycol)-b-poly(pentafluorophenyl methacrylate)/phosphonic acid and subsequently shell cross-linking with NH2-PEGNH2. The cross-linked PEG layer provided good water dispersity, nonfouling characteristics, and excellent long-term colloidal stability in phosphate-buffered saline in the range of 25−60 °C, whereas the high reactivity of the pentafluorophenyl ester with the amino group brought about ease of incorporation of functional moieties into LnNPs. Particularly, it was found for the first time that LnNPs with surface coating could endure the freeze-drying process without any sign of aggregation, which would not only greatly decrease the weight and storage and shipping room but also increase the storage shelf life with the preservation of their inherent properties, especially for LnNPs with some fragile bioconjugates while in solution. KEYWORDS: nanoparticle, lanthanide, block copolymer, surface coating, colloidal stability, lyophilization, nonfouling

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INTRODUCTION Lanthanide-containing nanoparticles (LnNPs) with unusual and unique magnetic and upconversion fluorescent properties have generated considerable interest and been widely employed as a versatile nanoplatform in biological labeling and imaging, disease diagnosis, and therapeutics.1−5 LnNPs are commonly prepared at high temperature in nonpolar solvents, such as oleic acid (OA), oleylamine (OM), and 1-octadecene, so as to yield nanoparticles coated with hydrophobic ligands, including OA or OM.1−5 Because the colloidal stability of LnNPs in aqueous media, especially in phosphate-buffered saline (PBS), which is commonly used to mimic human biological conditions, is one of the most important prerequisites to achieve satisfactory blood circulation time and biodistribution of nanoparticles, the surface of these LnNPs must be modified to render the LnNPs water-dispersible, while maintaining their colloidal stability and physiochemical properties. A variety of approaches are available to make LnNPs compatible with an aqueous environment. Coating of LnNPs with silica via an inverse microemulsion process is a popular method to obtain water dispersibility.6 Another approach is to encapsulate the LnNPs with amphiphilic polymers, such as poly(maleic anhydride-alt-1octadecene)-co-poly(ethylene glycol) and octylamine-grafted poly(acrylic acid) (PAA).7−9 The coating arose from hydro© 2017 American Chemical Society

phobic interactions between the hydrophobic block and OA on the surface of LnNPs, whereas the hydrophilic block rendered the nanoparticles water-soluble. Alternatively, one can carry out ligand-exchange reactions, replacing OA or OM ligands on the surface of the as-synthesized LnNPs with more hydrophilic polymer ligands, such as PAA, poly(N-vinylpyrrolidone), and methoxy-poly(ethylene glycol)-phosphonic acid (mPEGOPO3H2).10−14 Among these polymeric ligands, the PEGphosphate ligand is the most attractive one for two reasons: first, phosphates are known to be able to bind to Gd3+ (a wellknown rare earth element with magnetic properties) on the surface of LnNPs with greater affinities than carboxylic acid, which will prevent the exit of phosphate-based ligands from LnNPs to some extent and thus improve the colloidal stability of LnNPs; second, the outermost PEG layer endows the LnNPs with not only good water solubility and high colloidal stability but also excellent inhibition to nonspecific absorption of peptides and proteins. LnNPs coated with a PEG-based ligand with a monophosphonic acid anchoring group exhibited good water solubility and colloidal stability in water; however, they Received: January 30, 2017 Accepted: April 13, 2017 Published: April 13, 2017 14647

DOI: 10.1021/acsami.7b01452 ACS Appl. Mater. Interfaces 2017, 9, 14647−14655

Research Article

ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of the Preparation of Ultrastable LnNPs

of nanoparticles due to the enhanced attractive force.23 Works on the stability of silver and gold nanoparticles upon lyophilization from MacCuspie’s, Rotello’s, Alkilany’s, and Wang’s groups demonstrated that it was possible for silver and gold nanoparticles to by reconstituted with water or buffer solution with similar physiochemical and optical properties to those of the initial solution when the nanoparticles were coated with some suitable ligands.24−27 However, the surface chemistry of gold and silver nanoparticles is quite different from that of LnNPs, and these approaches might not be suitable for LnNPs. As far as we are aware, there is no report on the preservation of the colloidal stability of LnNPs upon lyophilization by surface coating, although various methods have been developed to improve the stability of LnNPs in different solutions. Herein, we reported a facile strategy to prepare highly colloidal stable and multifunctional LnNPs. First, the poly(ethylene glycol)-b-poly(pentafluorophenyl methacrylate) (PEG-b-PPFMA) amphiphilic diblock copolymer was synthesized as the ligand precursor, wherein the hydrophobic PPFMA block was used to introduce functional moieties (phosphonic acid, folic acid, and porphyrin) by virtue of its high reactivity toward the amino group and the PEG segment served as the hydrated layer to endow the nanoparticles with water solubility and nonfouling capacity. Subsequently, NaGdF4:Yb,Er nanoparticles with an average diameter of 15 nm, model LnNPs with upconversion fluorescence and magnetic properties, were coated with PEG-b-PPFMA/phosphonic acid by ligand exchange, followed by treatment with NH2-PEG-NH2 or PEG-NH2 (Scheme 1). The colloidal stability of these LnNPs in PBS buffer in the temperature range from 25 to 60 °C and over the freeze-drying process was examined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In addition, the antifouling property was investigated using bovine serum albumin (BSA) as a model protein, and folic acid and porphyrin were employed as model functional moieties to demonstrate the ease of surface functionalization.

showed limited or short-term stability in PBS as well as in alkaline solutions and tended to aggregate and even precipitate.15 Although the colloidal stability can be preserved by the addition of excess ligands, Zhao et al. reported that the presence of excess ligands would lead to unexpected etching of the nanoparticle.16 One strategy to enhance the stability is to increase the number of anchoring groups at the polymeric chain end by virtue of the multidentate coordination effect. For example, Gao et al. coated NaGdF4 nanoparticles with PEG-based ligands containing two phosphonic acid anchoring groups at the chain end.17,18 The nanoparticles stabilized by PEG with only one end phosphonic acid anchoring group quickly flocculated, whereas the nanoparticles coated by PEG possessing two end phosphonic acid anchoring groups showed excellent colloidal stability in PBS buffer, without aggregation.19−21 Cao et al. prepared a series of PEG-based ligands with one, two, and four phosphonic acid anchoring groups and systematically examined the colloidal stability of NaGdF4 stabilized by these PEG-based ligands in PBS buffer.22 Their results demonstrated that the colloidal stability was enhanced with an increase in the number of anchoring groups, as expected, and the nanoparticles coated with tetravalent PEGphosphonate-based ligands remained stable even in 200 mmol phosphate buffer to some extent.22 On the one hand, although multivalent PEG-phosphonate ligands can endow the nanoparticles with remarkable stability, their preparation was tedious and the number of anchoring groups was limited by the stepby-step synthesis strategy, even though a relatively efficient approach to prepare the tetravalent PEG-phosphonate ligand by a five-step synthesis method had been developed;16 moreover, the chemical inertness of PEG made further functionalization of the PEG ligand difficult to some extent. Lyophilization, a process to remove water from frozen solutions by sublimation, has been widely applied in pharmaceutical, food, and agricultural industries to preserve labile ingredients, increase their storage shelf life, and decrease their volume and weight in transportation.23 The freezing process would force the nanoparticles to have a small interparticle spacing, which normally results in the aggregation

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RESULTS AND DISCUSSION Preparation and Characterization of a PEG-Based Copolymer Ligand. First, we prepared the diblock copolymer 14648

DOI: 10.1021/acsami.7b01452 ACS Appl. Mater. Interfaces 2017, 9, 14647−14655

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to benzyl in the 1H NMR spectrum after deprotection (Figure 1B), distinctly verified the complete removal of benzyl to give phosphonic acid. It should be pointed out that no detectable peak attributed to pentafluorophenol in the 19F NMR spectrum (Figure S6) was observed over the deprotection, which indicated that the pentafluorophenyl ester moiety remained intact during the deprotection. In addition, the ratio of the chain length of PEG to that of the polymethacrylate-based segment is estimated to be 1.23 and 1.24 before and after the deprotection process, on the basis of the 1H NMR integration area ratio of typical protons, attributed to the PEG and polymethacrylate backbone, as seen in Figure 1A,B, respectively. This result shows that the polymeric structure remains intact, except for the deprotection of the phosphonate groups. Ligand Exchange to Prepare Copolymer-Coated Nanoparticles. Because of the unusual magnetic and upconversion fluorescent properties of NaGdF4:Yb,Er nanoparticles, they have been widely used as magnetic and fluorescent probes for biomedicinal imaging. In the present work, we prepared NaGdF4:Yb,Er nanoparticles according to previous reports17,18 and employed these nanoparticles as model LnNPs to examine their colloidal stability. As shown in Figure 2A, the as-prepared NaGdF4:Yb,Er nanoparticles were spherical. They had a number-average diameter of 15 ± 2.6 nm, obtained by measuring more than 200 nanoparticles in the TEM image, which was close to the intensity-averaged hydrodynamic diameter (Dh) of 21 nm obtained via DLS (Figure 2E). Before the ligand exchange, the NaGdF4:Yb,Er nanoparticles were first purified by precipitation with ethanol and three cycles of washing with a THF/ethanol mixture to remove excess OA, according to previous reports.17,18 Then, freshly purified NaGdF4:Yb,Er nanoparticles (10 mg) were dispersed in the THF solution (4 mL) of PEG-b-PPFMA/ phosphonic acid (7.5 mg/mL) for ligand exchange. Because phosphonic acid can bind more tightly to the surface of NaGdF4:Yb,Er nanoparticles than the carboxylic acid group of OA,30 the remaining OA on the surface of the NPs would be replaced by PEG-b-PPFMA/ phosphonic acid to afford the desired PEG-b-PPFMA-coated NaGdF4:Yb,Er nanoparticles (LnNPs@PEG-b-PPFMA). After the mixture was stirred in THF at 50 °C under a N2 atmosphere overnight for 24 h, the mixture was dialyzed against THF to remove excess ligands, assuming that the ligand-exchange reaction was complete. It should be pointed out that the size and morphology of nanoparticles observed by TEM were maintained after the ligand-exchange reaction and dialysis process (Figure 2B), which indicates that the etching of NPs did not occur. The etching proceeded in aqueous media with an excess of phosphate ligands,16 whereas in the current case, both ligand exchange and dialysis were performed in THF. A relatively low polarity of THF might be the reason for preventing nanoparticles from etching. The intensity-averaged Dh of LnNPs@PEG-b-PPFMA nanoparticles is 142 nm, with a polydispersity index (PDI) of 0.254 (Figure 2E, Table S1), after ligand exchange compared to 21 nm, with a PDI of 0.091, before ligand exchange. DLS also indicated a number-average diameter of 79 nm for LnNPs@ PEG-b-PPFMA nanoparticles from the same set of DLS data (Figure S7B), much smaller than the intensity-average value of 142 nm. We then performed TEM measurements on LnNPs@ PEG-b-PPFMA with and without staining and on LnNPs before and after ligand exchange to obtain the thickness of the polymer layer (Figures S8 and S9). The thickness of the

of PEG-b-PPFMA by RAFT polymerization using the PEGbased chain-transfer agent (PEG-CTA, Mn = 2250 g/mol, Mw/ Mn = 1.03), according to our previous report (Figures S1 and S2).28 The length of the PPFMA segment (NPFMA = 30) was evaluated from the 1H NMR spectrum (Figure S2B). Subsequently, potential anchoring groups of phosphate were installed into PEG-b-PPFMA by the amidation reaction between the pentafluorophenyl ester in the PPFMA segment and dibenzyl 3-aminopropylphosphonate to give PEG-bPPFMA/phosphate according to a previous report22 (Scheme S1 and Figure S3). The appearance of the typical peak at 36 ppm in the 31P NMR spectrum (Figure S4) of the product indicated the introduction of phosphonate moieties. The number of phosphonates in every PEG-b-PPFMA chain was also estimated from 1H NMR (Figure 1A), according to the

Figure 1. 1H NMR spectra of PEG-b-PPFMA/phosphonate (A) and PEG-b-PPFMA/ phosphonic acid (B) in CD2Cl2.

equation Nphosphonate = 45(Sd/10)/(Sc/4), in which Sd is the peak area of 10 protons of phenyl of dibenzyl phosphonate between 7.16 and 7.38 ppm, Sc is the peak area of four protons of OCH2CH2 of the PEG block at 3.60 ppm, and 45 is the number of ethylene glycol (EG) repeated units of the PEG segment. Furthermore, this reaction was monitored by 19F NMR (Figure S5A), wherein the number of phosphonate groups introduced into the PEG-b-PPFMA chain can be estimated from the amount of pentafluorophenol released from the PEG-b-PPFMA chain, assuming that all of the pentafluorophenols produced were the product of the amidation reaction between the pentafluorophenyl ester and dibenzyl 3aminopropylphosphonate. The average number of phosphonate groups in each PEG-b-PPFMA chain after the reaction is estimated to be 5, which was consistent with the result obtained from the 1H NMR spectrum in Figure 1A. This observation indicated that all free pentafluorophenols after the reaction were actually the product of the amidation reaction, that is, the pentafluorophenyl ester groups would not be hydrolyzed under the conditions for the amidation reaction. Then, phosphonate was deprotected into phosphonic acid in the presence of trimethylsilyl bromide to afford the target PEG-b-PPFMA/ phosphonic acid in dichloromethane.29 The disappearance of the resonance signal ranging from 7.16 to 7.38 ppm, attributed 14649

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Figure 2. TEM images of (A) pristine LnNPs, (B) LnNPs@PEG-b-PPFMA, (C) LnNPs@b-PEG, and (D) LnNPs@c-PEG. (E) CONTIN plots of the hydrodynamic distribution of 1.0 mg LnNPs/mL solutions of pristine LnNPs dispersed in THF, LnNPs@PEG-b-PPFMA dispersed in THF, LnNPs@b-PEG dispersed in water, and LnNPs@c-PEG dispersed in water. (F) TG curves of pristine LnNPs, LnNPs@PEG-b-PPFMA, LnNPs@bPEG, and LnNPs@c-PEG.

the surface of the nanoparticles were replaced by PEG-bPPFMA/phosphonic acid because of a higher affinity of phosphonic acid toward Gd3+ than that of carboxyl acid. Assuming that the mass loss of 41.0% originated from PEG-bPPFMA/phosphonic acid, the bulk density of the NPs in the inorganic core was 5.647 g/cm3 and the diameter of the spherical NPs was 15 nm, obtained from the TEM image, and the ligand density was estimated to be 0.5 ± 0.08 ligands/nm2, on the basis of the reported method for quantification of surface ligands on the NaYF4 nanoparticles.33 Subsequently, the shell of PEG-b-PPFMA was cross-linked with NH2-PEG-NH2 (Mn = 1000 g/mol), which was prepared according to a previous report,34 through amidation of the remaining pentafluorophenyl ester with amino groups of PEG to prepare cross-linked-PEG-coated NaGdF4:Yb,Er nanoparticles (LnNPs@c-PEG). To examine the effect of cross-linking

polymer layer in the dry state after ligand exchange is estimated to be about 5.5 and 8.5 nm from TEM and atomic force microscopy (AFM) measurements, respectively. In the current case, the Dh of LnNPs@PEG-b-PPFMA is surprisingly higher than both that of pristine LnNPs and their size in the dry state; even the higher sensitivity of DLS toward larger particles is taken into account.31,32 There is no unambiguous explanation for the observation, and the possible occurrence of aggregation of few LnNPs during the ligand-exchange process cannot be completely excluded, although the polymer-coated LnNPs seem to be well dispersed without obvious aggregation, as demonstrated by DLS, TEM, and AFM measurements. The mass loss in the range of 100 to 600 °C, attributed to ligands capping the surface of NPs, increased from 21.6 to 41.0% after the ligand-exchange reaction (Figure 2F). These observations clearly evidence that the pristine ligands of OA on 14650

DOI: 10.1021/acsami.7b01452 ACS Appl. Mater. Interfaces 2017, 9, 14647−14655

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ACS Applied Materials & Interfaces

Figure 3. CONTIN plots of the hydrodynamic distribution of 1.0 mg LnNPs/mL solutions of LnNPs@b-PEG (A) and LnNPs@c-PEG (B) in solutions with different pH values after 1 month.

Figure 4. CONTIN plots of the hydrodynamic distribution of 1.0 mg LnNPs/mL solutions of LnNPs@b-PEG (A) and LnNPs@c-PEG (B) in PBS buffer (10 mM) at different temperatures.

philic PEG segments. It could be observed that the wellseparated particles dispersed on TEM grids without any aggregation, as shown in Figure 2C,D. One could observe that both LnNPs@b-PEG and LnNPs@c-PEG in aqueous media have smaller intensity-average Dh’s than those of LnNPs@PEG-b-PPFMA in THF (Figure 2E). Although the attachment of PEG may make the outer layer more hydrated and further increase the size, different solvents were used in DLS measurements, which might lead to the difference between the intensity-average Dh of LnNPs@PEG-b-PPFMA in THF and that of LnNPs@b-PEG and LnNPs@c-PEG in aqueous media. Colloidal Stability of NPs with and without CrossLinking. Light scattering is a widely used tool to assess the colloidal stability of NPs by monitoring the change in the hydrodynamic diameter of NPs under different conditions.25−27 We began by examining the influence of the pH of the solution on the colloidal stability of the NPs. One could observe that the hydrodynamic diameter distributions of LnNPs@b-PEG and

on the stability of the NPs, monofunctional PEG-NH2 (Mn = 500 g/mol) was also employed in the amidation reaction instead of NH2-PEG-NH2 under similar conditions for preparing branched-PEG-coated NaGdF4:Yb,Er nanoparticles (LnNPs@b-PEG). After the amidation reaction, characteristic peaks of free pentafluorophenol appeared in the 19F NMR spectrum of the supernatant after centrifugation (Figure S10), which might indicate incorporation of the PEG segment into the shell of NaGdF4:Yb,Er. This observation was consistent with the increase in weight loss observed from thermogravimetric analysis measurements, wherein the weight loss in the range from 100 to 600 °C increased from 40.5 to 56.6 and 57.8% for the NPs treated with NH2-PEG-NH2 and PEG-NH2, respectively (Figure 2F). In addition, PEG-b-PPFMA-coated LnNPs were difficult to disperse in aqueous media and aggregates were formed during storage, whereas NH2-PEGNH2- and PEG-NH2-treated LnNPs@PEG-b-PPFMA could be easily dispersed in aqueous media without any sign of aggregation, which also indicated the incorporation of hydro14651

DOI: 10.1021/acsami.7b01452 ACS Appl. Mater. Interfaces 2017, 9, 14647−14655

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Figure 5. CONTIN plots of the hydrodynamic distribution of 1.0 mg LnNPs/mL solutions of LnNPs@b-PEG (A) and LnNPs@c-PEG (B) before and after lyophilization and LnNPs@b-PEG (C) and LnNPs@c-PEG (D) in an aqueous solution with BSA (2 mg/mL).

(Figure 4A, Table S3). These results demonstrate that the PEG-based ligand with five phosphonic acid groups and shell cross-linking can provide excellent colloidal stability to NPs in PBS buffer even at 60 °C, whereas the NPs coated with a PEGbased ligand without cross-linking flocculated to some extent. Colloidal Stability of NPs upon Lyophilization. Considering the high stabilities of LnNPs@b-PEG and LnNPs@c-PEG in PBS buffer even at a high temperature, we then tested whether PBS solutions of LnNPs@b-PEG and LnNPs@c-PEG were able to recover their original size distribution after lyophilization. The stability was examined by DLS to check for possible aggregation upon the freezedrying process for lyophilization. Satisfyingly, the dry powders of LnNPs@b-PEG and LnNPs@c-PEG obtained up on lyophilization can be resuspended as clear solutions after adding water and stirring for 30 min (Figure S11). DLS results showed that the size distributions of LnNPs@b-PEG and LnNPs@c-PEG suspended in the PBS buffer (10 mM) also overlapped with those before lyophilization, without any sign of aggregation (Figure 5A,B, Table S4). These observations clearly proved that the densely grafted PEG chains can serve as sterically bulky coatings on the surface so as to efficiently hinder the aggregation of nanoparticles during the lyophilization process, regardless of whether the layer was cross-linked or

LnNPs@c-PEG, with peaks at 91.3 nm, remained unimodal with almost no change or sign of aggregation in solutions with pH values of 3.0, 7.0, and 9.0, even after storage for 1 month (Figure 3, Table S2). We then tested the colloidal stability of LnNPs@b-PEG and LnNPs@c-PEG in PBS buffer at room temperature. The hydrodynamic diameter distribution remained unimodal, without any shift to a larger size or appearance of a peak of a larger size over 1 month (Figure 4). Considering the temperature of the human body (∼37 °C) and possible temperature fluctuation during storage and transportation, we also examined the thermal colloidal stabilities of LnNPs@b-PEG and LnNPs@c-PEG in PBS buffer over the temperature range from 25 to 60 °C. Samples were incubated in PBS for 0.5 h at each temperature (25, 30, 40, 50, and 60 °C) prior to measurements. For LnNPs@c-PEG, they still retained the colloidal stability, as indicated by the retention of the size and size distribution after 1 month (Table S3), whereas for LnNPs@b-PEG without cross-linking, the peak value shifted from 90.6 nm (PDI = 0.228) to 122 nm (PDI = 0.222) (Table S3). We found that the colloidal stability of LnNPs@c-PEG could be preserved even at 60 °C for 1 day without any aggregation (Figure 4B, Table S3), whereas some flocculation occurred for LnNPs@b-PEG at 60 °C after storage for 1 day 14652

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PO3(Ph)2/folic acid chain, which was consistent with the result obtained from UV/vis spectroscopy. Subsequently, porphyrin was also introduced by an amidation reaction between the pentafluorophenyl ester and 5,10,15,20-tetrakis(4-aminophenyl) porphyrin. The appearance of the characteristic absorption peaks of prophyrin in the UV/vis spectrum of the obtained LnNPs also demonstrated the incorporation of porphyrin (Figure S13). The content of porphyrin was estimated to be 0.4 per chain on the basis of the extinction coefficient of porphyrin (1.84 × 105 M−1 cm−1) in Figure S14C and the UV absorbance of the PEG-b-PPFMA/PO3(Ph)2/folic acid/porphyrin copolymer (Figure S14D). Then, the surface of NPs was coated with PEG-b-PPFMA/PO3H2/folic acid/ porphyrin under similar conditions for the ligand-exchange reaction, followed by shell cross-linking with NH2-PEG-NH2. Well-separated particles, without any aggregation, dispersed on the TEM grid were observed (Figure S15A), along with a narrow size distribution and an average hydrodynamic diameter of 91.3 nm obtained from DLS measurements (Figure S15B). These results demonstrated that PEG-b-PPFMA/PO3H2/folic acid/porphyrin-coated NPs had good dispersity in aqueous media. In addition, the colloidal stability of PEG-b-PPFMA/ PO3H2/folic acid/porphyrin-coated NPs in PBS buffer was accessed by DLS (Figure S16). The hydrodynamic diameter distribution of PEG-b-PPFMA/PO3H2/folic acid/porphyrincoated NPs in PBS buffer after storage for 7 days almost overlapped with that before storage. This observation confirmed that aggregation did not occur even if functional moieties of folic acid and porphyrin were introduced onto their surfaces.

not. Previous investigations on the stability of PEG-coated polymeric and inorganic nanoparticles showed that although the PEG layer could endow nanoparticles with a high colloidal stability in aqueous solutions, these nanoparticles usually would aggregate upon lyophilization, probably due to the intra-/ interparticular bridges formed upon crystallization of the PEG domains during freeze drying.23,35 Therefore, additional cryoprotectants are needed to maintain colloidal stability over lyophilization. An’s work on comblike PEG-coated nanogel showed that brushlike PEG chains tethered on the surface of the nanogel might hamper the crystallization of PEG segments and thus allow the nanogel to survive lyophilization without any aggregation.36 In our current work, the nanoparticles were also covered with comblike PEG chains, and the highly diverse conformations of the comblike PEG chains were able to impede the crystallization of PEG domains, resulting in colloidal stability of the NPs against the freeze-drying process for lyophilization. Undesirable absorption of proteins onto the surface of nanoparticles is one of the prerequisites for their applications in vitro and in vivo. PEG is a kind of FDA-approved biocompatible polymer with a high nonfouling property. We then examined the stabilities of LnNPs@b-PEG and LnNPs@cPEG in aqueous media with the addition of BSA by DLS. The peak at about 10 nm originated from BSA, and the peak at about 100 nm was attributed to LnNPs@b-PEG and LnNPs@ c-PEG, as shown in Figure 5C,D. Both LnNPs@b-PEG and LnNPs@c-PEG remained without any sign of flocculation after 1 week. The high stability of LnNPs@b-PEG and LnNPs@cPEG showed that the highly dense PEG chains on the surface of LnNPs can efficiently prevent the absorption of proteins onto their surface. Surface Functionalization via the Amidation Reaction. The amidation reaction between the pentafluorophenyl ester and amino group is considered to be a “click reaction” because of its high efficiency under mild conditions. One of the attractive advantages of the currently employed method for the surface coating of NPs with the PPFMA-containing copolymer is the ease of introduction of various functionalities. We performed the proof-of-concept experiment by incorporating of folic acid,37 a widely used and high-affinity ligand of folate receptors overexpressed by numerous cancer cells for targeted drug delivery and imaging, and porphyrin,38 a popular potential drug for imaging and photodynamic therapy. We first synthesized amino-containing folic acid according to a previous report39 (details of the synthesis and characterization can be found in the Supporting Information). Then, folic acid functionalities were incorporated into PEG-b-PPFMA/phosphonate to give PEG-b-PPFMA/phosphonate/folic acid. The incorporation of folic acid was confirmed by the release of pentafluorophenol (Figure S5B), appearance of characteristic peaks originating from folic acid in the 1H NMR spectrum of PEG-b-PPFMA/PO3(Ph)2/folic acid (Figure S12), and the typical absorption of folic acid in the UV/vis spectrum (Figure S13). The extinction coefficient of folic acid at 358 nm (ε358) was obtained from the UV/vis absorbance standard curve (Figure S14A), with a value of 6.19 × 103 M−1 cm−1; therefore, the content of folic acid in the copolymer was evaluated to be 3.6 folic acids per chain via UV/vis spectroscopy (Figure S14B) and ε 358 . On the basis of the content of released pentafluorophenol, the content of folic acid was also estimated from 19F NMR (Figure S5B), and it was found that there were about 3.6 folic acid molecules in every PEG-b-PPFMA/

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CONCLUSIONS We reported a coating strategy to prepare highly stable LnNPs using the PEG-b-PPFMA diblock copolymer, wherein the outermost PEG layer provided LnNPs with water dispersity and nonfouling properties and the inner layer of the PPFMA segment was employed for the installation of not only anchoring groups of phosphonic acid and functional moieties but also monoamino or diamino PEG chains for increasing their colloidal stabilities via an efficient amidation reaction between the amino group and the pentafluorophenyl ester of the PPFMA segment. Both shell-cross-linked and non-crosslinked NPs demonstrated long-term stability in PBS buffer over 1 month, without aggregation, over temperatures ranging from 25 to 50 °C. Some aggregation occurred for non-cross-linked NPs at 60 °C, whereas no sign of aggregation was observed for the cross-linked NPs. These NPs, regardless of whether the shell was cross-linked or not, exhibited an excellent capacity against freeze drying or lyophilization and can redisperse into aqueous media after stirring for 30 min, without aggregation. These NPs can be further functionalized to introduce folic acid and porphyrin by taking advantage of the high reactivity of the pentafluorophenyl ester toward the amino group. Given the high stability, especially the capacity against freeze drying for lyophilization, nonfouling property, and ease of surface functionalization of the obtained LnNPs, our strategy for surface coating of LnNPs could be of potential interest for broader application of LnNP-based nanoplatforms.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01452. 14653

DOI: 10.1021/acsami.7b01452 ACS Appl. Mater. Interfaces 2017, 9, 14647−14655

Research Article

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Experimental details for polymer synthesis; ligand exchange; stability assessment of nanoparticles; gel permeation chromatography; 19F, 31P, and 1H NMR spectra of the obtained copolymers; TEM, AFM, and DLS of aqueous solutions of the obtained nanoparticles (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-21-54925606. Fax: +86-21-664166128 (C.F.). *E-mail: [email protected]. Tel: +86-21-54925310. Fax: +86-21-64166128 (X.H.). ORCID

Xiaoyu Huang: 0000-0002-9781-972X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Basic Research Program of China (2015CB931900), the National Key R&D Program of China (2016YFA0202900), the National Natural Science Foundation of China (51373196, 21504102, and 21632009), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB20000000), the Youth Innovation Promotion Association of Chinese Academy of Sciences (2016233), and the Shanghai Scientific and Technological Innovation Project (14JC1493400, 16JC1402500, 16520710300, and 14520720100). We would like to thank Dr. Yi Hou of the Institute of Chemistry, Chinese Academy of Sciences, for the preparation of NaGdF4:Yb,Er nanoparticles.

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DOI: 10.1021/acsami.7b01452 ACS Appl. Mater. Interfaces 2017, 9, 14647−14655