Cisplatin-Encapsulated Polymeric Nanoparticles with Molecular

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Biological and Medical Applications of Materials and Interfaces

Cisplatin-encapsulated Polymeric Nanoparticles with Molecular Geometry-regulated Colloidal Properties and Controlled Drug Release Yun-Ho Jeong, Hyeon-Woo Shin, Ji-Yeong Kwon, and Sang-Min Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06905 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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ACS Applied Materials & Interfaces

Cisplatin-encapsulated Polymeric Nanoparticles with Molecular Geometry-regulated Colloidal Properties and Controlled Drug Release Yun-Ho Jeong, Hyeon-Woo Shin, Ji-Yeong Kwon, and Sang-Min Lee* Department of Chemistry, The Catholic University of Korea, Bucheon, Gyeonggi-do 14662, Korea. KEYWORDS: Block-copolymers, Cisplatin, Colloidal Micelles, Nanoparticles, Drug delivery, pH-sensitive drug release

ABSTRACT

Encapsulation of chemotherapeutic agents inside a nanoscale delivery platform can provide an attractive therapeutic strategy with many pharmaceutical benefits, such as increased plasma solubility, prolonged in vivo circulation, and reduced acute toxicity. Given that the biological activities of polymeric nanoparticles are highly dependent on their colloidal structures, the molecular geometry-regulated programming of self-assembled nanoscale architecture is of great interest for chemical design of an ideal delivery platform. In this report, we demonstrate the molecular geometry of block-copolymer excipients can govern the level of drug-loading capacity

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and core hydrophobicity of polymeric nanoparticles, which can eventually control the pHsensitive drug release property.

Atom-transfer radical polymerization was employed as a

controlled synthetic method for the copolymer excipients, which contain the metal-chelating poly(acrylic acid) block linked to either a small mPEG-grafted poly(methacrylate) to generate a bulky brush-like chains or a simple linear mPEG segment. During the coordination of cisdiammineplatinum(II) as an active pharmacophore of cisplatin, aqueous-phase SEC analyses exhibited highly different self-association kinetic regimes prompted by versatile molecular geometry of copolymer excipients, which further allows us to explore the molecular geometry−colloidal property relationship.

Introduction Cisplatin, a Platinum (II)-based inorganic cytotoxic agent, has long been employed as a clinically available small-molecule anticancer drug.1-3

To reduce the systemic toxicity of

cisplatin, numerous attempts have been made to encapsulate this cytotoxic agent inside versatile delivery platforms such as lipid vesicles4-5 and polymeric micelles.6-8 Specifically, amphiphilic block copolymer-based micelle systems, containing drug-loaded hydrophobic core surrounded by hydrophilic corona, are of great interest for the encapsulation and targeted delivery of many cisplatin analogs9-10 due to the facile synthetic modularity and stimuli-responsive triggered property of modern polymer materials.11-13

Hence, the controlled release of cisplatin

pharmacophore (referred to here as PtII) from a smart delivery platform can reduce the acute toxicity, while the nanoscale encapsulation can provide additional benefits such as enhanced


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adsorption to tumor tissue, prolonged half-life for the plasma circulation, and protection of drug molecules from a premature degradation and clearance.14-17 One of the facile preparation strategies for PtII-encapsulated micellar system includes exploiting the PtII-mediated self-assembly of block-copolymers, which contain the metalcoordinating ligand block and micelle stabilizing poly(ethylene glycol) (PEG) chains.18 In this approach, divalent PtII conjugation induces a chain contraction of metal-bound blocks,19 resulting in the nanoscale self-aggregation of PtII-bound copolymers to minimize the surface energy, which eventually leads to the formation of polymeric nanoparticles with core/corona structure. Hence, the simple mixing of PtII drugs with various metal-coordinating block-copolymers allows for the one-pot synthesis of PtII-encapsulated polymeric nanoparticles in aqueous solution. However, despite such facile preparation method, major drawbacks of PtII-encapsulated polymeric nanoparticles include the lack of an efficient stimuli-responsive trigger for tunable drug-release, which requires a chemical incorporation of specific functional groups such as acidcleavable linkers.20 Given that the biological activities of conventional drug delivery platforms are highly dependent on their colloidal properties,21-22 several physicochemical parameters regarding the colloidal structures have been investigated to understand the rational relationship between the molecular geometry of polymeric components and the resulting colloidal property.23-26 In this context, many studies have been focused on the critical micelle concentration (cmc) as a representative indicator for the thermodynamic stability of colloidal micelles, demonstrating high dependency of cmc on the hydrophobicity and crystallinity of core block.27 Additionally, the grafting density of corona chains also plays an important role on the colloidal structure28 and biological stability against protein or carbohydrate adsorption.29-30 Despite the precedent studies,


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however, tailoring of colloidal structures and properties via synthetic modulation of molecular geometry has been mostly focused on the conventional micelle systems based on the blockcopolymers of intrinsic amphiphilic characters.31-32 Herein, we demonstrate the molecular geometry-dependent variation of colloidal structures and their relationship to kinetic properties for both PtII-mediated self-association of blockcopolymers and drug release profiles. To this end, core/corona-type PtII-encapsulated polymeric nanoparticles (PtII-PNPs) with various molecular geometries were prepared with modularly synthesized block-copolymers, containing metal-binding poly(acrylic acid) block joined either a small mPEG-grafted poly(methacrylate), poly(PEGMA), to generate a brush-type structure or a simple linear methoxy-terminated PEG (mPEG) segment to compare the different types of PEGylation at the surface of PtII-PNPs. Depending on the molecular geometry of corona chains, kinetically different reaction regimes were observed during the PtII-mediated self-association process, which exhibited highly inert PtII-PNPs with bulky poly(PEGMA) chains, while relatively labile structures were obtained with linear mPEG chains at corona. Such different kinetic properties of each nanoparticle for the self-association process determined the final PtIIencapsulated colloidal structure, which leads to the highly varied PtII loading amounts with different core-packing density and hydrophobicity, significantly affecting on the pH-sensitive drug release profiles. Additionally, molecular geometry-dependent colloidal features, including the aggregation number of unimers and surface grafting density, were also investigated, which can provide a chemical basis for designing a suitable copolymer excipient on demand for the required colloidal activity.


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Results and discussion Synthesis and Characterization of Block-copolymer Excipients. To prepare the molecular geometry-modulated polymeric nanoparticles encapsulating cisplatin pharmacophores, two sets of block-copolymers have been synthesized using a known atom-transfer radical polymerization method33 (Scheme 1 and Figures S1−S2 in SI): (1) block-copolymers containing a linear PAA block of constant molecular weight (4 kDa) and a brush-type small mPEG-grafted poly(methacrylate), poly(PEGMA), with various chain lengths (PAA-b-PEGMA) and (2) typical block-copolymers containing PAA of two different molecular weights (4 and 7 kDa) and a linear mPEG (2 kDa) block (PAA-b-PEG). For the preparation of PAA-b-PEGMA, poly[tert-butyl acrylate] (PtBA) has been initially polymerized with ethyl α-bromoisobutyrate (EBiB) initiator, followed by the subsequent polymerization of PEGMA as a second block. For PAA-b-PEG syntheses, α-bromoisobutyryl bromide (BiBB) was initially modified with linear mPEG2k to prepare a macroinitiator mPEG-BiB,34 which was then subjected to the polymerization of tertbutyl acrylate (tBA) to provide PAA-b-PEG after the acid-mediated deprotection (acidolysis) of tert-butyl groups in PtBA. For further systematic investigation, we have prepared a series of PAA-b-PEGMA and PAA-b-PEG copolymers, containing various lengths of neutral PEGMA and anionic PAA blocks (Table 1).

Scheme 1. Synthesis of brush-like PAA-b-PEGMA and linear PAA-b-PEG block-copolymers via atom-transfer radical polymerization.


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Table 1. Characterization of PAA-b-PEGMA and PAA-b-PEG Copolymer Excipients. Mn (g/mol) b entry

Block-copolymer excipients a

notation PAA

PEG/ PEGMA

χPAA χPEG (wt%)c (wt%)c

electrokinetic mobility (µe) (10−9 m2/V·s) d

1

PAA60-b-PEGMA30 4k-g-15k 4 000

15 000

21

79

−29 ± 2

2

PAA60-b-PEGMA20 4k-g-10k 4 000

10 000

29

71

−43 ± 3

3

PAA60-b-PEGMA4

4k-g-2k

4 000

2 000

67

33

−51 ± 2

4

PAA60-b-PEG45

4k-l-2k

4 000

2 000

67

33

−33 ± 3

5

PAA100-b-PEG45

7k-l-2k

7 000

2 000

78

22

−38 ± 3

a

subscribed numbers indicate the final degree of polymerization obtained from 1H NMR.

b

measured by 1H NMR.

c

relative weight fraction of each polymer block.

d

electrokinetic mobility monitored by 0.2 wt% sample solution at pH 7.4 and 25 °C.

Given that the copolymers contain both anionic and neutral blocks in a single chain with various molecular geometry, the electrokinetic mobilities (µe) of each copolymer have been measured in 10 mM phosphate buffer (pH 7.4) to examine the characteristic molecular geometry-


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dependent diffusion behaviors in aqueous solution. As expected from the anionic nature of PAA block, highly negative µe has been observed for all the as-prepared copolymers, while the µe values of each copolymer were substantially varied, depending on the relative weight fraction of anionic PAA blocks and the molecular geometry of mPEG/PEGMA blocks within a copolymer chain. For PAA-b-PEGMA, the polymers with short PEGMA block (high χPAA) exhibited more negative µe (entry 1−3 in Table 1), because of the increased negative charge density within a polymer chain and the reduced hydrodynamic volume of neutral PEGMA block.35 Notably, despite the identical χPAA value, the copolymers containing linear mPEG (4k-l-2k, entry 4 in Table 1) exhibited less negative µe value (−33 × 10-9 m2/V·s), compared against 4k-g-2k (−51 × 10-9 m2/V·s, entry 3 in Table 1), due to the relatively expanded hydrodynamic volume of linear mPEG chain.36 Hence, it can be postulated that both the weight fraction and the molecular geometry of each block can significantly modify the charge-induced mobility of copolymer chains, further providing a molecular basis of diffusion property for the subsequent PtII-mediated self-association process.

Fabrication and SEC Analyses of Colloidal PtII-PNPs. For the formation of PtII-conjugated polymeric nanoparticles (PtII-PNPs), cisplatin pharmacophore, cis-[Pt(NH3)2(H2O)2]2+ (PtII), was added to the pH-adjusted copolymer solution (pH 7.0) and agitated for 48 h at 25 °C (Figure 1A). Given that the binding of divalent metal ions has been known to induce a conformational change of acrylate-based polyelectrolytes,19 coordination of PtII on the PAA blocks can facilitate the self-association of copolymer chains with PtII-bound PAA blocks aggregated at the core, surrounded by the hydrophilic mPEG/PEGMA chains.18 This structural feature of PtII-PNPs can be verified by 1H NMR analyses in D2O (Figure S3 in SI). When an equimolar amount of PtII


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was added to the copolymer solution of D2O, a selective suppression of proton signal occurs at the PtII-bound PAA residues by hydrophobic segregation, while the proton peaks from mPEG/PEGMA blocks remained essentially unchanged from those of parent PtII-free copolymers via substantial hydration in aqueous solution, suggesting the PtII-mediated formation of micelle-like core/corona-type colloidal nanostructures.

Figure 1. (A) Schematic illustration of self-association process for the formation of PtIIPNPs. (B) Aqueous-phase SEC chromatograms of 4k-g-15k copolymer before (black dotted line) and after PtII coordination (red solid line), using 10-mM phosphate-buffered solution (pH 7.4, 140 mM NaNO3) as an aqueous eluent. To verify the separation capability of SEC, an incomplete reaction mixture of PtII and 4k-g-15k was analyzed for PtII-PNPs. The PtII amount in SEC eluate was estimated by SnII-mediated colorimetric analysis37 and plotted as a chromatogram at bottom (blue line). (C) Hydrodynamic diameter profiles of SEC eluates at the VR of 16.5 (top), 18.0 (middle), and 20.0 mL (bottom). Each fraction analyzed by DLS is indicated by red, blue, and green arrows on red chromatogram in Figure 1B.


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The formation of PtII-PNPs was further studied by aqueous-phase size-exclusion chromatography (SEC) analyses, employing 10-mM phosphate-buffered solution (pH 7.4, 140 mM NaNO3) as an aqueous eluent, where the nanoscale aggregates can be discriminated from the free polymer chains by hydrodynamic volume change.38 More than 0.1 M of salt has been introduced in the aqueous eluent to shield the possible ionic interactions of anionic copolymers during the SEC analyses, while the minimal effect of nitrate ions on the PtII-bound PAA complex was expected due to the weakly binding nature of nitrate as a coordinating ligand, compared against the relatively robust coordination of carboxylates to PtII ions.39 Figure 1B shows a representative SEC chromatogram obtained from the mixture of PtII and 4k-g-15k after 6 h agitation. Compared to the native copolymer, which was eluted as a single peak at the retention volume (VR) of ~20 mL (black dotted chromatogram), the PtII/4k-g-15k mixture exhibited additional peaks at VR ≈ 16−19 mL (red solid chromatogram), showing an increased hydrodynamic volume of polymer chains by PtII-mediated association. Additionally, co-elution of PtII at VR ≈ 16−19 mL (blue chromatogram) also supported the successful incorporation of PtII pharmacophores within the copolymer aggregates. When the SEC eluates were analyzed by dynamic light scattering (DLS), distinct scattering signals from the fractions at VR ≈ 16.5 and 18.0 mL exhibited an extensive nanoscale aggregation of PtII-bound copolymers with an average hydrodynamic diameter (DH) of 43 ± 10 and 34 ± 7 nm, respectively (Figure 1C). Notably, the SEC chromatogram revealed that a significant amount of native copolymers remained without PtII-conjugation (green arrow at VR ≈ 20 mL in Figure 1B and negligible DLS signal in Figure 1C, bottom), while the unreacted PtII species eluted at VR ≈ 28−29 mL.

Additionally,

unassociated PtII-bound unimers were also observed as a small hump at VR ≈ 21−22 mL possibly due to the reduced hydrodynamic volume of PtII-bound PAA blocks through the metal-mediated


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conformational changes.19

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This aspect can be attributed to the insufficient self-association

efficiency of PtII-bound 4k-g-15k, presumably because of the bulky PEGMA block in the copolymer chains,40 which requires an extended reaction time for further association (vide infra). Together with the DLS data, these results demonstrate that the clear monitoring of PtIImediated association of copolymers in aqueous solution was made possible by SEC, which also allows for the successful separation of nanoparticles from the residual PtII ions, native copolymer chains, and unassociated PtII-bound unimers (Figure S4 in SI). Based on the aforementioned SEC separation method, we endeavor to analyze the temporal peak evolution of nanoparticles as a function of agitation time to understand the PtII-mediated association process.

During the SEC measurements, the residual reactive PtII ions can be

removed by the phosphate-containing aqueous eluent because of the known binding property of phosphate toward diamminediaquo PtII complex,39, 41 while the PtII-incorporated nanoparticles can be monitored without significant perturbation as supported by the intact structural integrity of PtII-PNPs under our aqueous eluent condition (Figure S5 in SI). As such, the chromatograms of PtII/copolymer mixtures with 4k-g-2k, 4k-l-2k, and 7k-l-2k excipients have been obtained in a timely manner and the PtII-mediated association processes of each copolymer were analyzed to understand the effect of molecular geometry and size of copolymers on the PtII-mediated association process. For 4k-g-2k, the native copolymer chains were initially eluted at VR ≈ 21 mL (black arrow in Figure 2A), while a new peak for PtII-PNPs begins to appear at VR ≈ 17 mL after 1 min agitation of PtII/copolymer mixture (red arrow). After 5 min agitation, the peak intensity from nanoparticles gradually increased equivalent to that of the free unimers, showing significant amount of unimer transfer to PtII-PNPs during this period. Such unimer consumption has been completed within ~10 min, as observed by the substantially attenuated unimer peak


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intensity. Interestingly, the final VR of PtII-PNPs increased to ~17.8 mL, indicating that the size of PtII-PNPs slightly decreased after 15 min agitation. Similar results have been observed at the SEC chromatograms of 4k-l-2k and 7k-l-2k, while more rapid unimer consumption has been monitored for both copolymers (Figure 2B, C). Presumably, such rapid association process of PAA-b-PEG can be ascribed to the flexible structure of linear mPEG block, which allows for the facile unimer incorporation into the cores of nuclei.42 Notably, 7k-l-2k containing the elongated PAA block exhibited even faster association of PtII-bound polymer chains, followed by the unimer consumption completed within ~2 min. Such labile association of 7k-l-2k can be attributed to the highly enhanced hydrophobicity of the elongated PAA blocks upon the PtII coordination.

Figure 2.

Agitation time-dependent evolution of SEC chromatogram by PtII-mediated

nanoparticle formation from the copolymer (A) 4k-g-2k, (B) 4k-l-2k, and (C) 7k-l-2k.

The strong dependency of unimer association rates on the molecular geometry of copolymer excipients was clearly observed, when the temporal changes of the nanoparticle peak intensity was plotted as a function of agitation time (Figure 3A). Indeed, the labile PAA-b-PEG excipients exhibited the rapid completion of nanoparticle formation process, while PAA-b-


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PEGMA required significantly extended agitation time to reach the stable nanostructures in equilibrium. We attributed such extended association time of PAA-b-PEGMA to the bulky PEGMA chains, which can not only enhance the aqueous solubility of PtII-bound unimers but also sterically retard the unimer incorporation into the nuclei, eventually leading to the slow formation of PtII-PNPs.

Figure 3. The agitation time-dependent evolution of (A) nanoparticle peak intensity and (B) the apparent mass change of PtII-PNPs monitored by SEC chromatogram. The apparent mass of each nanoparticle was obtained from the calibration curve prepared with PEG standards (Figure S2B in SI). The time-dependent SEC analyses further demonstrate that the PtII-mediated nanoparticle formation follows the conventional micellization process of typical amphiphilic blockcopolymers in aqueous medium, where the initial nucleation occurs via rapid unimer association, followed by the relatively slow redistribution of micelle population by unimer exchange or micelle fusion/fission.42 In contrast to the fast micellization of conventional block-copolymer amphiphiles, which typically undergoes in µsec scale, however, relatively slow formation of PtIIPAA complexes leads to the steady generation of amphiphilic PtII-bound unimers and hence, allowing for the temporal monitoring of PtII-mediated unimer association by SEC


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analyses. Once the sufficient amount of PtII ions binds to the acrylate residues, the increased hydrophobicity of PtII-bound PAA residues induces the extensive association of unimers at early stage,

where

the

size

of

the

initial

nuclei

rapidly

increases

with

broad

size

distribution. Subsequently, as the significant amount of PtII-bound unimers are consumed, the steady redistribution of nanoparticle population by unimer exchanges leads to the narrow size distribution with the average mass of PtII-PNPs slightly decreased (vide infra).43 The aforementioned two-step formation process of PtII-PNPs can be clearly observed when the agitation time-dependent apparent mass changes of nanoparticles were examined by comparing to the SEC calibration curve of PEG standards (Figure 3B). Consistent with our postulation, the temporal analysis of apparent mass change revealed the mass-increasing initial stage, which is completed within several min, followed by the size-redistribution stage, where PtII-PNPs reach the stable structures via the equilibration of mass transfer between the nanoparticles. Indeed, in conjunction with the agitation time-dependent unimer association shown in Figure 3A, the relatively labile copolymer excipients, such as those containing the linear mPEG or small PEGMA block, exhibited the rapid stabilization in the mass changes of PtII-PNPs, while PAA-bPEGMA copolymers with bulky PEGMA block demonstrated a slow and steady increase in the mass of nanoparticle (Figure S6 in SI), further verifying the inert nature of 4k-g-15k excipient for PtII-mediated association.

Core/Corona Structures of Colloidal PtII-PNPs. After the purification of excess PtII ions by filter centrifugation, the substantially neutralized zeta potential of PtII-PNPs at pH 7.4 verified the successful formation of well-defined core/corona-type nanoparticles with maximum PtII incorporation (Figure S7 in SI). Further physicochemical characterization was then carried out


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to evaluate the colloidal structures of the resulting PtII-PNPs (Table 2). The initial DLS measurements at pH 7.4 exhibited the low polydispersity indices (PI) of nanoparticles, allowing for the cumulant analyses of PtII-PNPs (Figure S8 in SI). As expected, the elongation of PEGMA block in PAA-b-PEGMA increased the number-based hydrodynamic diameters (DH) of PtII-PNPs, from 29 ± 8 nm for 4k-g-2k, which contains the short PEGMA block, to 39 ± 11 nm and 44 ± 12 nm for 4k-g-10k and 4k-g-15k, respectively (entry 1–3 in Table 2). In contrast, PAA-b-PEG exhibited negligible changes in DH of PtII-PNPs upon the elongation of coreforming PAA block (DH = 23 ± 6 nm for 4k-l-2k and 25 ± 4 nm for 7k-l-2k) (entry 4,5 in Table 2). Transmission electron microscope (TEM) observation also validated the DLS results, further demonstrating the spherical morphology of PtII-PNPs (Figure 4A–E). In each image, highly contrasted core was observed at the center of nanoparticles, exhibiting the electron-rich PtII species localized inside the particles, while the surrounding mPEG/PEGMA chains were barely visible due to the relatively low electron density of soft polymer chains.44 Table 2. Physicochemical Characterization of Colloidal PtII-PNPs. entry

copolymer excipients

DH (nm)a

Dc (nm)b

Lcrn (nm)c

1

4k-g-15k

44 ± 13

20 ± 3

12

104

390

31.1

33

1.58

2

4k-g-10k

39 ± 11

18 ± 2

10

100

280

27.5

48

0.85

3

4k-g-2k

25 ± 6

17 ± 2

4

42.5

240

26.4

230

0.19

4

4k-l-2k

23 ± 6

13 ± 1

5

48.8

107

20.2

268

0.05

5

7k-l-2k

25 ± 4

14 ± 2

5.5

84.0

80

13.0

177

0.23

Vcrn Nagge (nm3)d

dgraft cmc 2 2 f (/10 nm ) (mg/L)g

Kv (×105)h

a

number-based hydrodynamic diameter (DH) determined by DLS (Figure S8 in SI).

b

diameter of PtII-incorporated core measured by TEM observation (Figure S9 in SI).

c

thickness of hydrophilic corona layer on the surface of PtII-PNPs determined from DH and Dc.

d

estimated volume of single corona chain determined from DH and Nagg.


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e

estimated average aggregation number of PtII-bound unimers per single particle.

f

estimated grafting density of corona chain per 100 nm2 at the core/corona interface.

g

critical micelle concentration determined by pyrene excitation spectra in DI water at room temperature (Figure S10A–E in SI). h

apparent partition equilibrium constant of pyrene probe (Figure S10F in SI).

Figure 4. TEM images of PtII-PNPs prepared from copolymers (A) 4k-g-15k, (B) 4k-g-10k, (C) 4k-g-2k, (D) 4k-l-2k, and (E) 7k-l-2k. (F) Average diameter of PtII-incorporated core (Dc) of PtII-PNPs measured from the corresponding TEM images (Figure S9 in SI).

TEM observation further allows for the estimation of both the size of PtII-incorporated core (Dc) and the thickness of surrounding corona layer (Lcrn) by comparing to the corresponding DH values (Table 2). As expected from the constant length of core-forming PAA blocks in PAA-b-


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PEGMA, only small changes were observed in the Dc values of PtII-PNPs (Figure 4F).45 Instead, Lcrn values significantly changed for each sample of PtII-PNPs: as the molecular weight of PEGMA block increases from 2k to 10k and 15k, Lcrn of the resulting nanoparticles increased from 4 to 10 and 12 nm (entry 1–3 in Table 2), indicating that the overall DH variation of PtIIPNPs predominantly arises from the corona thickness change of PEGMA layer while the sizes of the PtII-incorporated cores being relatively constant (Figure 5A).46 For PAA-b-PEG, apparently similar colloidal structures of PtII-PNPs were observed for 4k-l-2k and 7k-l-2k with only minute differences in Dc and Lcrn (entry 4, 5 in Table 2). Presumably, PtII-mediated cross-linking of PAA blocks may contract the PtII-incorporated core,47 which can counterbalance the significant size increase of core by the elongated PAA block of 7k-l-2k.

Further estimation of

hydrodynamic volume occupied by individual corona chain (Vcrn) revealed remarkably different surface structures on PtII-PNPs. Because Vcrn is highly susceptible to the polymer grafting density as well as the surface curvature of nanoparticles,29 Vcrn can provide more precise information on the polymeric structures grafted on the surface of nanoparticles. Indeed, 7k-l-2k exhibited much higher Vcrn (84.0 nm3) than those from 4k-g-2k and 4k-l-2k (Vcrn = 42.5 nm3 and 48.8 nm3, respectively), showing highly expanded volume of individual corona chains on the surface of nanoparticles (Figure 5A).


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Figure 5. (A) Schematic illustration for the molecular geometry-dependent colloidal structures of PtII-PNPs, demonstrating the different core-packing density with various thickness of corona layer and grafting density. (B) Comparison of aggregation number (Nagg) of polymer chains in a single particle and the grafting density (dgraft) of polymer chains at the core/corona interface of PtII-PNPs. (C) Comparison of critical micelle concentration (cmc) of PtII-PNPs and the corresponding partition equilibrium constant (Kv) of pyrene probe.

The different structures of corona chains on PtII-PNPs can be understood by mean aggregation number of unimers in a single particle (Nagg) and the chain grafting density (dgraft) at corona, both of which were highly varied, depending on the molecular geometry of the parent copolymer excipients (Figure 5B). Consistent with the labile nature of PAA-b-PEG as shown in the


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previous section of this paper, 7k-l-2k exhibited the low values of Nagg and dgraft, which can lead to the loosely packed cores and the expanded volume of corona chain to stabilize the hydrophobic cores in aqueous solution. In contrast, the elongation of PEGMA block in PAA-bPEGMA increased both Nagg and dgraft for the resulting PtII-PNPs (entry 1–3 in Table 2 and Figure 5B). This aspect can be attributed to the relatively inert nature of PAA-b-PEGMA, which allows for the extended association time for the PtII-bound unimer incorporation. Indeed, similar results have been reported previously with a micelle-type nanoparticle composed of amphiphilic copolymers, which exhibited an increased aggregation number as the PEG grafting numbers at the polymer backbone increased.48 Therefore, given the similar core sizes observed for each nanoparticle, the increased Nagg for PAA-b-PEGMA with elongated PEGMA block indicates an enhanced packing of PtII-bound PAA at the core, as shown by the high dgraft value, further suggesting the formation of robust nanostructures with high core-packing density.

Validation of Micelle Property by Pyrene Study. Given the physical estimation of core/corona structures have been successfully achieved with PtII-PNPs, the critical micelle concentration (cmc) of each nanoparticle was examined with pyrene fluorescence probe to understand the dynamic micellar properties. To this end, we employed a cmc measurement method at room temperature by comparing the excitation intensity ratio of pyrene at 339 and 334 nm49 (I339/I334, Table 2 and Figure S10A–E in SI) in salt-free aqueous condition (pH 7.0) to monitor the colloidal integrity of PtII-PNPs without the effect of salt.50 Among the PtII-PNPs with PAA-b-PEGMA, 4k-g-15k exhibited the lowest cmc (33 mg/L), possibly attributed to the effective shielding of hydrophobic cores as well as the enhanced solubilization by the bulky PEGMA chains at corona.28,

48

Consistently, the reduction of PEGMA fraction in parent


18

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copolymers increased the cmc of PtII-PNPs (entry 1–3 in Table 2). These results have also been verified by Nile red, another well-known fluorophore for cmc determination,51 under the same condition (Figure S11 in SI). For PAA-b-PEG, 7k-l-2k exhibited lower cmc (177 mg/L) than that of 4k-l-2k (268 mg/L), attributed to the elongated core-forming PAA block in 7k-l-2k, which is consistent to the colloidal features previously reported with the amphiphilic polymer micelles.27 Given

the

molecular

association

property

of

pyrene

toward

the

hydrophobic

microenvironments, the partition equilibrium constant (Kv) of pyrene can provide additional information on the relative hydrophobicity of PtII-incorporated cores.49 Hence, Kv values of each nanoparticle have been obtained by the linear regression of excitation intensity ratios against the polymer concentration (Table 2 and Figure S10F in SI). As expected from the robust colloidal structure with elongated PEGMA block, 4k-g-15k exhibited the highest Kv value, indicating the enhanced hydrophobicity at the core of PtII-PNPs. Conversely, the low Kv values observed from the nanoparticles with short PEGMA supported the loosely packed PtII-bound PAA blocks inside PtII-PNPs, demonstrating the inverse relationship of Kv to the cmc of each nanoparticle (Figure 5C). For PAA-b-PEG, the high fraction of core-forming PAA blocks in 7k-l-2k induced relatively more compact association of unimers with enhanced hydrophobicity, compared to those from 4k-l-2k, as shown by the low cmc and high Kv values (entry 4, 5 in Table 2). Taken together, it can be postulated that the molecular geometry of parent copolymer excipients can dictate the colloidal core/corona structure and the resulting micellar property. Hence, high fraction of hydrophilic PEGMA block induces the relatively slow PtIImediated unimer association in aqueous media and leads to the nanoparticles of robust structures with high Nagg and low cmc by the strong association of unimers at the cores. In contrast, the


19


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reduction of the PEGMA fraction in the copolymer excipients facilitates the hydrophobic association of PtII-bound unimers, resulting in the rapid formation of PtII-PNPs with low Nagg and loosely packed core, where the unimers can easily dissociate at low concentration. On the other hand, the elongation of core-forming PAA block in PAA-b-PEG induced the strong association of PtII-bound unimers, showing the enhanced packing of PtII-PAA residues at the core as shown by the high Kv and low cmc. This aspect of PtII-PNPs further enables the colloidal structuredependent PtII-release kinetics under acidic condition (see the next section).

Table 3. PtII-loading quantity, drug-leakage after lyophilization, and pH-dependent drug-release kinetics of PtII-PNPs. entry

copolymer excipients

YPt (mol%)a

NPt (PtII/particle)b

fPt (wt%)c

DHLyo (nm)d

Lk.Lyo kpH 7.4 kpH 4.0 (mol%)e (h−1)f (h−1)f

1

4k-g-15k

75

17 600

35

51 ± 14

4.0

0.03

0.7

2

4k-g-10k

73

14 300

44

45 ± 11

4.1

0.11

1.0

3

4k-g-2k

72

9 500

61

33 ± 13

4.2

0.15

1.8

4

4k-l-2k

53

3 100

53

35 ± 13

6.6

0.29

3.0

5

7k-l-2k

51

4 100

57

100 ± 30

6.9

0.18

2.4

a

PtII encapsulation yield, compared to the PtII initially added for the nanoparticle formation.

b

estimated number of PtII ions encapsulated in a particle.

c

weight fraction of PtII pharmacophore inside PtII-PNPs.

d

hydrodynamic diameter of PtII-PNPs after lyophilization/rehydration. (Figure S12 in SI)

e

PtII leakages after lyophilization/rehydration, compared to the PtII initially loaded in PtIIPNPs. f

pH-dependent apparent drug-release rates at 37 °C in either pH 7.4 or pH 4.0 buffer, containing 150 mM NaCl (Figure 7).


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Quantitative Estimation of PtII Encapsulated in PtII-PNPs. Given that the physicochemical analyses of colloidal structures have been accomplished with PtII-PNPs, the amount of PtII pharmacophore encapsulated inside the nanoparticles was quantitatively examined before the evaluation of drug-release profiles under various chemical condition. Because the equimolar amount of PtII, compared to the acrylate units in each copolymer, has been conjugated for the construction of PtII-PNPs, final PtII encapsulation yield (YPt) can be obtained by comparing the PtII loaded inside nanoparticles to the initial feeding amount after the purification of unloaded PtII species. Remarkably, more than 70 mol% YPt has been achieved with PtII-PNPs composed of PAA-b-PEGMA, showing 72, 73, and 75 mol% YPt for 4k-g-2k, 4k-g-10k, and 4k-g-15k, respectively (entry 1–3 in Table 3). This can be possibly attributed to the inert nature of PAA-bPEGMA for the PtII-mediated nanoparticle formation, allowing for the extended reaction time for PtII conjugation (Figure 6A). In comparison, PtII-PNPs with PAA-b-PEG exhibited slightly lower YPt with 53 and 51 mol% for 4k-l-2k and 7k-l-2k, respectively (entry 4, 5 in Table 3), providing comparable values to the previously reported PtII loading yields inside polymeric nanoparticles.52 Presumably, the rapid completion of PtII-mediated association with labile PAAb-PEG allows only a short period of time for PtII conjugation, eventually leading to the relatively low YPt for the PtII encapsulation (Figure 6A).


21


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Figure 6. Colloidal property changes of PtII-PNPs, depending on the molecular geometry of copolymer excipients. (A) PtII encapsulation yields (YPt) of each copolymer excipient, compared with PtII-mediated unimer association time as measured by the aqueous-phase SEC analyses. (B) Hydrodynamic diameter changes (DHLyo) and PtII-leakages from PtII-PNPs after lyophilization and rehydration. (C) Apparent turbidity changes of PtII-PNPs after incubation with bovine serum albumin (BSA) for 12 and 36 h at 37 °C. The solution turbidity was monitored by UV−Vis transmission at 480 nm. The turbidity changes of BSA/cisplatin mixture and native BSA solution were also monitored as positive and negative controls, respectively. Asterisks represent significant differences compared against the corresponding 4k-g-15k samples.

Statistical

significance was determined by one-way ANOVA, followed by Tukey’s post-hoc analysis (p < 0.05).

For further evaluation of drug loading property, the absolute quantity of PtII pharmacophore encapsulated inside a single particle (NPt) has been estimated, using the previously observed Nagg and YPt values. For PtII-PNPs with 4k-g-2k, approximately 9,500 PtII ions were encapsulated inside a single nanoparticle. Despite the constant PAA block lengths in PAA-b-PEGMA, NPt increased to ~17,600 PtII ions per particle as the length of PEGMA block increases to 15 kDa (entry 1–3 in Table 3), presumably attributed to the increased Nagg with longer PEGMA block. This aspect is consistent with the enhanced packing of PtII-bound PAA blocks at the core for 4k-g-15k as observed by the high Kv value in Figure 5C. Not surprisingly, 4k-l-2k exhibited the lowest NPt (~3,100 PtII ions) as expected from the low values of Nagg and YPt, also supporting the loosely packed core with attenuated hydrophobicity inside nanoparticles with sparsely grafted unimer chains. In stark contrast to NPt values, the relative weight fraction of PtII


22

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pharmacophore inside PtII-PNPs (fPt) was highly varied for each nanoparticle, depending on the molar mass of parent copolymers: For PAA-b-PEGMA, the fPt values substantially decreased with the elongated PEGMA block (entry 1–3 in Table 3) due to the increased portion of PEGMA chains in a particle. On the other hand, the elongation of PAA block in PAA-b-PEG induces only slight change in fPt (entry 4, 5 in Table 3), compensated by the decreased Nagg for 7k-l-2k, demonstrating a strong dependency of fPt on the lengths of both blocks.

Colloidal Stability against Lyophilization and Protein-induced Aggregation. For an extended shelf life, many pharmaceutical products can be dehydrated by lyophilization (freezedrying) to prevent the hydrolytic/enzymatic degradation before long-term storage. However, the lyophilized colloidal particles have often suffered from the insufficient stability, showing significant aggregation or precipitation after rehydration. Hence, versatile saccharides or PEGbased polymers have been employed as an effective lyoprotectant.48, 53 As such, to monitor the colloidal stability against lyophilization/rehydration (lyostability), the aqueous solution of PtIIPNPs (10 mM phosphate buffer, pH 7.4 with 150 mM NaNO3) was first freeze-dried without any additives and rehydrated by deionized water, which were then subjected to the evaluation of both drug leakage (Lk.Lyo) and hydrodynamic diameter change (DHLyo). Remarkably, PtII-PNPs with PAA-b-PEGMA exhibited an excellent lyostability with only minute changes in DHLyo and negligible PtII leakages (~4 mol%) after lyophilization/rehydration (entry 1–3 in Table 3). Presumably, the bulky PEGMA layer at the corona of PtII-PNPs prevents the inter-particle contact of PtII-incorporated cores by steric repulsion, preserving the integrity of core/corona structures under dehydrated condition.


23


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In stark contrast, PtII-PNPs prepared with PAA-b-PEG exhibited approximately 150% and 400% changes in DHLyo for 4k-l-2k and 7k-l-2k, respectively (Figure 6B). Despite the relatively expanded volume of mPEG layer as observed by high Vcrn in Table 2, the attenuated lyostability of 7k-l-2k can arise from the low grafting density (dgraft) of copolymer chains on the nanoparticles, resulting in the insufficient passivation of hydrophobic cores during the freezedrying process and the irreversible inter-particle aggregation after rehydration. However, relatively low Lk.Lyo values (6.6 and 6.9 mol% of PtII leakages for 4k-l-2k and 7k-l-2k, respectively) indicate that most PtII pharmacophores are still bound to the copolymer excipients despite the significant aggregation after lyophilization/rehydration. These observations suggest that the colloidal lyostability can indeed be acquired through the compact surface passivation by bulky hydrophilic polymers with high grafting density at the corona of nanoparticles. Also supporting the critical role of corona chains on the colloidal stability is the proteininduced aggregation assays, which can demonstrate the molecular geometry-dependent colloidal stability. To this end, PtII-PNPs were incubated with bovine serum albumin (BSA, 50 mg/mL) in a 10-mM phosphate-buffered saline (pH 7.4, 150 mM NaCl) at 37 °C as a virtual blood plasma condition with a model serum protein.29 After 12 and 36 h incubation, the solution turbidity changes of each nanoparticle solution were monitored through the comparison of UV−Vis transmittance at 480 nm and plotted as a bar graph (Figure 6C). Because serum albumin is the most abundant protein in blood, which provides the binding sites for cisplatin adsorption during the in vivo circulation,54 molecular cisplatin induced a significant aggregation via PtII-mediated cross-linking of proteins. In contrast, as expected from the excellent surface passivation property of PEG-based polymers,55 most PtII-PNPs significantly prevented PtII-mediated protein aggregation, while slightly increased turbidity observed with 7k-l-2k after 36 h. This aspect is


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consistent with the attenuated lyostability of 7k-l-2k, which leads to the formation of PtII-PNPs with sparsely grafted mPEG chains at corona, demonstrating the important role of corona chains with high grafting density on the colloidal stability of nanoparticles.

pH-responsive Drug-release Kinetics of PtII-PNPs. Given that the kinetically different regimes for the PtII-mediated unimer association have been observed, we endeavor to evaluate the molecular geometry-dependent drug-release kinetics of PtII-PNPs as a reverse process of PtIImediated unimer association. As the enhanced drug-release property was previously observed for the versatile PtII-coordinated polymeric nanoparticles under acidic condition,47,

56

similar

acid-sensitive PtII-release property was expected with our PtII-PNPs. Hence, the temporal release of PtII pharmacophore from PtII-PNPs was monitored in a timely manner during the dialysis of nanoparticles against either pH 4.0- or pH 7.4-buffered solution with 150 mM NaCl at 37 °C. The apparent PtII release rate constants (k) were then estimated by the linear regression of initial 48 h-release data (Table 3), assuming the early stage of release process as a pseudo-first order reaction.13, 57 As expected from the hydrophobic nature of PtII-coordinated cores, which can substantially prevent the ion-mediated PtII dissociation, all PtII-PNPs were relatively inert at pH 7.4, showing only ~10 mol% of PtII leakages after 48 h with kpH 7.4 of less than 0.3 h−1 (Figure 7A,B).


25


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Figure 7. pH-dependent release profiles of PtII pharmacophore from PtII-PNPs prepared with (A) PAA-b-PEGMA and (B) PAA-b-PEG excipients at 37 °C. The PtII release profiles were monitored at either pH 4.0 (solid lines) or pH 7.4 (dashed lines). As a control, free PtII ions dialyzed without any polymeric excipients were rapidly released out of the membrane within a few minutes at pH 7.4. (C) pH-dependent apparent PtII release rate constants monitored at either pH 4.0 (purple bars) or pH 7.4 (gray bars). Also shown is the apparent Kv values observed by the pyrene study (red line), showing the inverse relationship between the acid-triggered PtII release rates and the core hydrophobicity of PtII-PNPs.

When the nanoparticles were incubated under acidic condition, the PtII release rates (kpH 4.0) were remarkably enhanced for both PAA-b-PEGMA and PAA-b-PEG, exhibiting more than 10fold increase in kpH

4.0,

compared to the corresponding kpH

significant enhancement in kpH

4.0

7.4

values (Table 3). Notably, a

from PAA-b-PEG was observed over those from PAA-b-

PEGMA, showing a near-complete release of PtII after 60 h with 82 and 91% of PtII release for 7k-l-2k and 4k-l-2k, respectively (Figure 7B). As hypothesized previously, this enhanced PtII release can be attributed to the labile property of PAA-b-PEG excipients and the relatively low PtII packing density at the core, which allows for the enhanced permeation of proton ions, eventually leading to the accelerated dissociation of PtII from the PAA residues under acidic condition.44 In stark contrast, PAA-b-PEGMA exhibited high variation in final PtII-release amounts, depending on the length of PEGMA block. As the molecular weight of PEGMA block decreased from 15k to 10k and 2k, an increase in kpH 4.0 has been observed from 0.7 to 1.0 and 1.8 h−1 (entry 1–3 in Table 3) with 30, 54, and 81% of final PtII release observed, respectively (Figure 7A). This is consistent with the reduced hydrophobicity of PtII-incorporated cores as


26

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previously shown by the attenuated Kv values with decreased fraction of PEGMA block, further demonstrating the inverse relationship between the kpH

4.0

and Kv values (Figure

7C). Additionally, after certain amount of PtII release, the formation of hydrogen bonds between the grafted PEG segments and protonated PAA blocks under acidic condition can significantly retard the further release process,58-59 eventually showing the stable plateau after ~60 h. Together with the aforementioned colloidal structures of PtII-PNPs, this result suggests that the PtII release kinetics from PtII-PNPs can indeed be dictated by the molecular geometry of parent copolymer excipients, which can also modulate the colloidal structures of the resulting nanoparticles.

Conclusion In conclusion, we have demonstrated a facile regulation strategy for the modulation of colloidal structure and drug release property with a PtII-encapsulated delivery system based on the block-copolymer excipients with versatile molecular geometry. Depending on the relative fractions between the PtII-binding PAA residues and the hydrophilic mPEG/PEGMA block, the copolymer excipients exhibited remarkably different kinetic regimes for the PtII-mediated unimer association. By the facile regulation of molecular geometry for the copolymer excipients, the resulting PtII-PNPs exhibited versatile tunability on final loading amount of PtII pharmacophore, the compactness and hydrophobicity of PtII-incorporated core, and the grafting density of corona chains on the surface of nanoparticles. As demonstrated here, this aspect additionally leads to the tunable PtII-release property under acidic condition as well as the enhanced colloidal stability against lyophilization and protein adsorption by the efficient surface passivation of hydrophobic core. The delicate controllability of core/corona structures and the relevant functional features in


27


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our PtII-PNPs, which is made possible entirely by the synthetic tunability of copolymer excipients in a molecular scale, will further allow for the efficient delivery and the enhanced potency of versatile PtII-based pharmacophores to the disease sites.

Experimental Section Materials. Poly(ethylene glycol) methyl ether (mPEG, average Mn = 2000 g/mol), ethyl αbromoisobutyrate (EBiB, 98%), α-bromoisobutyryl bromide (BiBB, 98%), copper(I) bromide (98%), N,N,N′,N′′,N′′-Pentamethyldiethylenetriamine (PMDETA, 98%), cis-diamminedichloroplatinum (II) (cisplatin >99.9%, trace metals basis), bovine serum albumin (BSA, 96%), PEG standards for SEC calibration, and Nile red (technical grade, 90%) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. tert-butyl acrylate (tBA, 98%) was stirred over CaH2 under nitrogen and fractionated by vacuum transfer right before use. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 500 g/mol) were purified by passing through an alkaline aluminum oxide column to remove the inhibitors. Tin (II) chloride dihydrate

(SnCl2·2H2O),

triethylamine

(TEA),

4-(dimethylamino)pyridine

(DMAP),

trifluoroacetic acid (TFA, 99%), hydrochloric acid (HCl, 35%), and all other reagents including typical organic solvents were purchased from Samchun Chemical Co. (Pyeong-Taek, Korea) and used as received. Regenerated Cellulose (RC) dialysis membrane (3.5 kDa MWCO) and SlideA-LyzerTM Dialysis Cassette G2 (3.5 kDa MWCO, 3 mL) were purchased from Spectrum Laboratories (Thermo scientific, Waltham, MA). Deionized (DI) water was obtained from Human Power I+ Scholar-UV (Human Corporation, Seoul, Korea) (18.2 MΩ cm resistivity).


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Measurements. Instrumental analyses were carried out at the Cooperative Center for Research Facilities of the Catholic University of Korea. Fourier-transformed nuclear magnetic resonance (NMR) spectroscopy was carried out on an AVANCE III 300 MHz spectrometer. Chemical shifts of 1H NMR spectra are reported in ppm against residual solvent resonance as an internal standard (CDCl3 = 7.27 ppm, D2O = 4.8 ppm). The samples were prepared by dissolving 5 ± 1 mg in 0.7 mL of deuterated solvents in a 5 mm NMR tube. Ultraviolet−visible (UV−Vis) absorption spectra were obtained on a Lambda 35 spectrophotometer (Perkin Elmer, Waltham, MA).

Fluorescence emission/excitation spectra were obtained on a FluoroMate FS-2

fluorometer (Scinco Co., Ltd. Seoul, Korea) at a scan speed of 100 nm/min (λex = 336 nm, λem = 392 nm, slit width = 2.5 nm for pyrene; λex = 550 nm, λem = 644 nm, slit width = 5 nm for Nile red). PtII concentration of PtII-PNPs determined by SnII-mediated colorimetric quantification was verified with an iCAP Q inductively coupled plasma-mass spectrometer (ICP-MS, Thermo Fisher Scientific, Waltham, MA) after dissolution of samples in aqua regia. Dynamic light scattering (DLS) and zeta (ζ) potential measurements were carried out with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK), which uses a 633 nm He−Ne laser with the noninvasive backscatter method (detection at 173° scattering angle). Correlation data were fitted by the method of cumulants to obtain the translational diffusion coefficient (Dt), from which the hydrodynamic diameters (DH) of the nanoparticles were calculated by the Stokes−Einstein equation. The size distribution of the nanoparticles was obtained by the nonnegative least-squares (NNLS) analysis. The ζ-potential of each sample was calculated from the electrophoretic mobility values using the Smoluchowski equation. Unless noted otherwise, all samples were dispersed in 10 mM phosphate buffer (pH 7.4) for measurements. The data reported represent an average of ten measurements with five scans each.


29


Page 30 of 45

Energy-filtering transmission electron microscope (EF-TEM) observation was carried out with a LIBRA 120 (Carl Zeiss, Oberkochen, Germany) with a beam voltage of 120 kV and a slowscan charge-coupled device (CCD) at National Instrumentation Center for Environmental Management in Seoul National University. The sample solution (50 µL) was placed on a copper grid (200 mesh) for 30 s, and the excess solution was blotted with filter paper. The grid was dried at room temperature for 24 h. Size-Exclusion Chromatography (SEC) Analysis. Aqueous-phase SEC analysis was carried out on an Agilent 1260 Infinity LC equipped with three Shodex polyhydroxymethacrylate gel columns (SB-802.5HQ, SB-803HQ, and SB-804HQ columns) in series including OHpak SB-G guard column, which is connected to an Agilent 1260 RI detector. 10-mM phosphate-buffered solution (pH 7.4, 140 mM NaNO3) was used as an aqueous eluent at a flow rate of 0.5 mL/min. The SEC instrument was calibrated with six different linear PEG/PEO standards (0.55, 2, 5, 35, 100, and 600 kDa) and the apparent mass of each sample was automatically calculated by OpenLAB CDS ChemStation (ver. A.2.3.57) software. The obtained calibration curve was plotted in Figure S2B in SI. Syntheses of Poly(acrylic acid)-b-(Poly ethylene glycol methacrylate) Block-copolymer (PAA-b-PEGMA). PAA-b-PEGMA block-copolymer was prepared using a modified literature procedure.33 Briefly, CuBr (41.6 mg, 0.29 mmol) were added to a 50-mL dry Schlenk flask equipped with a magnetic stir-bar, which was then sealed with a rubber septum, degassed and back-filled with nitrogen three times. Anhydrous acetone (5 mL), tert-BA (4.2 mL, 29 mmol), and PMDETA (60 µL, 0.29 mmol) were subsequently added via syringe under inert condition. Next, the solution was stirred until the Cu complex was formed, which can be easily recognized by color change from colorless/turbid to greenish clear solution. After complex formation, ethyl


30

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α-bromoisobutyrate (43 µL, 0.29 mmol) was added and degassed by three cycles of freezepump-thaw. Then, the flask was placed in an oil bath and polymerization was carried out at 60 °C. After the predetermined reaction time, the polymerization was quenched by placing the reaction flask in an ice bath. After the evaporation of acetone under reduced pressure, the reaction product was purified by passing through a neutral alumina column with THF to remove the copper catalysts.

The resulting product was precipitated with a 10-fold excess of

water/MeOH (50/50 vol/vol) solution. The precipitate was dried under vacuum and obtained as a colorless product. For the polymerization of second block, bromo-poly(tBA) (Mn = 7900, 3.1 g, 0.39 mmol), CuBr (56.0 mg, 0.39 mmol), and anhydrous acetone (5 mL) were added to a 50-mL dry Schlenk flask equipped with a magnetic stir-bar. PEGMA (7.8 g, 15.6 mmol) and PMDETA (81 µL, 0.39 mmol) were subsequently added via a purged syringe, which was then stirred until the Cu complex was formed. The mixture was degassed by three cycles of freeze-pump-thaw. Then, the flask was placed in an oil bath and polymerization was carried out at 60 °C. After the predetermined reaction time, the polymerization was quenched by placing the reaction flask in an ice bath. After the evaporation of acetone under reduced pressure, the reaction product was purified by passing through a neutral alumina column with THF to remove the copper catalysts. For acid-mediated deprotection of tert-butyl groups in poly(tBA), the polymer product (8.7 g) was refluxed in 1,4-dioxane (60 mL) containing 10-mL HCl at 85 °C for 6 hours. After cooling down to room temperature, the crude product was neutralized with aqueous NaOH (15 mL, 10%) and dialyzed (MWCO = 3500 Da) against DI water (7 × 1000 mL) for 7 days with water change in every 24 h. After dialysis, the product was dried by lyophilization and obtained as a colorless product.


31


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Syntheses of mPEG-modified Macroinitiator (mPEG-BiB). mPEG-modified macroinitiator was prepared using a modified literature procedure.34 Briefly, to a 100-mL three-neck roundbottom flask equipped with a magnetic stir-bar was added poly(ethylene glycol) methyl ether (mPEG, 2.0 g, 1.0 mmol), TEA (2.54 mL, 18 mmol), DMAP (9.5 mg, 0.078 mmol), and anhydrous THF (23 mL).

After the reaction mixture was cooled in an ice-water bath, 2-

bromoisobutyryl bromide (0.25 mL, 2.0 mmol) dissolved in 7 mL THF was added dropwise to the reaction flask for 1 h at 0 °C under inert condition, which was then kept at 0 °C for additional 3 h. Next, the reaction mixture was stirred at room temperature for 48 h during which mPEGmodified macroinitiator was formed. After the reaction mixture was concentrated under reduced pressure, the product was precipitated from the reaction mixture by addition of an excess amount of cold diethyl ether. The precipitated product was collected by filtration and dried at room temperature under vacuum for 24 h. Syntheses of Poly(acrylic acid)-b-Poly(ethylene glycol) Block-copolymer (PAA-b-PEG). mPEG-BiB macroinitiator (1.85 g, 0.86 mmol) and CuBr (0.12 g, 0.86 mmol) were added to a 50-mL dry Schlenk flask equipped with a magnetic stir-bar. The flask was sealed with a rubber septum, degassed and back-filled with nitrogen three times. To the reaction flask was added anhydrous acetone (20 mL), tert-BA (18.9 mL, 0.129 mol), and PMDETA (0.18 mL, 0.86 mmol) under inert condition, which was then stirred until the Cu complex was formed. The mixture was degassed by three cycles of freeze-pump-thaw. Then, the flask was placed in an oil bath and polymerization was carried out at 60 °C.

After the predetermined reaction time, the

polymerization was quenched by placing the reaction flask in an ice bath. After the evaporation of acetone under reduced pressure, the reaction product was purified by passing through a neutral alumina column with THF to remove the copper catalysts. For acid-mediated deprotection of


32

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tert-butyl groups in poly(tBA), to a 250-mL round-bottom flask equipped with a magnetic stirbar was added the polymer product (8.4 g), DCM (80 mL), and TFA (10 mL, 99 %), followed by stirring for 72 h at room temperature. After DCM was evaporated under vacuum, the crude product was neutralized with aqueous NaOH (10 mL, 10%) and dialyzed (MWCO = 3500 Da) against DI water (7 × 1000 mL) for 7 days with water change in every 24 h. After dialysis, the product was dried by lyophilization and obtained as a colorless product. Preparation of PtII-conjugated Polymeric Nanoparticles (PtII-PNPs). To a 50-mL roundbottom flask equipped with a magnetic stir bar was added cisplatin (40 mg, 133 µmol) dissolved in DI water (18 mL) at 50 °C. To the resulting solution was added silver nitrate (41 mg, 240 µmol, 1.8 equiv) with a trace amount of nitric acid, and the reaction mixture was allowed to stir for 72 h at room temperature in the dark. To completely precipitate out the slowly forming silver chloride, the solution was stored at 4 °C overnight. After silver chloride precipitate was removed by centrifugal filter (15 mL, MWCO = 10 kDa), followed by additional purification by syringe filter (pore size = 0.2 µm), the aqueous solution of cis-diamminediaquo PtII complex was obtained (12 mM, pH ~5.0). The PtII pharmacophore was next subjected to the PtII-encapsulated polymeric nanoparticle fabrication process with block-copolymer excipients.

In a typical

procedure, to a 5-mL glass vial, containing the pH-adjusted aqueous solution (pH 7.0, 1.2 mL) of PAA4k-b-PEGMA15k excipient (5 mg, 0.26 µmol) was added the equimolar amount of PtII (12 mM, 1.3 mL) and incubated at 25 °C for 48 h. The resulting PtII-PNPs were purified by centrifugal filter (5 mL, MWCO = 10 kDa), followed by redispersion in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaNO3 for physiologically isotonic condition. The final product was obtained in 2 mg/mL of polymer concentration. Addition of 150 mM NaNO3 during the nanoparticle formation exhibited apparently 10−20% increase in the final size of nanoparticles


33


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albeit with the weak binding property of nitrate to PtII ions (Figure S4 in SI), while the nanoparticle formation was significantly prohibited in 150-mM phosphate-buffered solution by the strong binding property of phosphate to PtII ions.39, 41 Physicochemical Estimation of Colloidal Parameters. The diameter of PtII-incorporated core (Dc) was visually estimated from the TEM images (Figure S9 in SI). The aggregation number of unimers (Nagg) in a single particle was obtained from the following equation:60

=

(4/3)

where Rcore = core radius; VPAA = volume of acrylate repeat unit in PtII-bound PAA (0.170 nm3), based on the reported hydration radius of cis-diammineplatinum(II) complex in aqueous solution (r = 2.32 Å)61 and the known molecular volume of acrylic acid (0.114 nm3); NPAA = degree of polymerization of the PAA block. The hydrodynamic volume of single corona chain (Vcrn) on the core/corona interface of PtIIPNPs was calculated using the formula:

=

−

where VPNP = hydrodynamic volume of PtII-PNPs obtained from DLS data; Vcore = volume of PtII-coordinated core estimated from Dc. The grafting density of corona chain per 100 nm2 at the core/corona interface (dgraft) was obtained from the following equation:29


34

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=

× 100 nm 4

Determination of Critical Micelle Concentration (cmc) of PtII-PNPs.

Critical micelles

concentration (cmc) of PtII-PNPs was determined in DI water using a standard pyrene procedure.49 Briefly, aliquots of pyrene (10 µL) from a stock solution in acetone (6.0 × 10 −5 M) were added to 5-mL vials and acetone was removed by evaporation. To each vial was added the aqueous solutions of PtII-PNPs (1.0 mL) with various concentrations and agitated at room temperature overnight to equilibrate the pyrene/PtII-PNPs mixture. The final concentration of pyrene in each sample was 6.0 × 10

−7

M. The excitation spectrum ranged from 300 to 350 nm

was obtained by monitoring the emission at λem = 392 nm and the excitation intensity ratio at 339 and 334 nm (I339/I334) was plotted as a function of polymer concentration to determine the cmc of each sample.

The pyrene partition equilibrium constant (Kv) was obtained from the following

equation: [Py]& ( − (&) ,- . / = = [Py]' (*+ − ( 10000 where χcore = weight fraction of core block; c = concentration of polymer; ρcore = density of core, which we assume to be same as the value of cisplatin (3.7 g/mL). For verification of pyrene results, the cmc of PtII-PNPs prepared with 4k-g-15k and 4k-g-2k was additionally determined with Nile red, following the modified literature procedure.51 Briefly, aliquots of Nile red (45 µL) from a stock solution in ethanol (2.8 × 10 −5 M) were added to 5-mL vials and ethanol was removed by evaporation. To each vial was added the aqueous solutions of PtII-PNPs (1.0 mL) with predetermined concentrations and agitated at room temperature overnight to equilibrate the Nile red/PtII-PNPs mixture. The final concentration of


35


Page 36 of 45

Nile red in each sample was 1.25 µM. The fluorescent emission intensity at 644 nm was observed by excitation at λex = 550 nm. The cmc of each nanoparticle was determined by the inflection point at the plot of emission intensity against polymer concentration (Figure S11 in SI). Determination of PtII Quantity in PtII-PNPs. The amount of PtII pharmacophore in PtII-PNPs was determined using a modified literature procedure.37 Briefly, an aliquot of PtII-PNPs (200 µL) was added to aqueous HCl (2 M, 50 µL) and incubated for >3 h to completely release the PtII ions and convert them into cisplatin. Then, the quantity of PtII ions was determined by colorimetric analysis with 10% SnCl2−HCl (2 N) solution with UV−Vis spectroscopy, using the predetermined molar extinction coefficient (ε) of the Sn−Pt complex (5610.8 M−1 cm−1 at λmax = 405 nm), which was obtained from the PtII standard solution and the calibration curve (Figure S13 in SI). The calibration curve was verified with ICP-MS for further quantification. The number of PtII ions encapsulated in a particle (NPt) was obtained from the following equation: = × 11 × 2 The weight fraction of PtII pharmacophore inside PtII-PNPs (fPt) was obtained from the following equation:

3 =

/1 × 4 ( /1 × 4 ) + ( /1 × 4 67& )

where NA = Avogadro number; MPt = molecular weight of PtII pharmacophore; Mpolymer = molecular weight of polymer.


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Colloidal Stability Test against Lyophilization. An aliquot of PtII-PNPs solution (1.2 mL, 1 mg/mL) dispersed in 10-mM phosphate buffer (pH 7.4, 150 mM NaNO3) in a 1.7-mL microtube was frozen by liquid nitrogen and dried under vacuum. The resulting freeze-dried powder was then reconstituted with DI water (1.2 mL) by vigorous vortexing for 10 min. The mean DH changes and PtII-leakages from the rehydrated PtII-PNPs were determined by DLS and SnCl2mediated colorimetric analysis after purification of leaked PtII outside the nanoparticles by centrifugal filtration. Colloidal Stability Test against Protein-induced Aggregation. An aliquot of PtII-PNPs solution (1.5 mL) was mixed with 10-mM phosphate-buffered saline (pH 7.4 and 150 mM NaCl) containing bovine serum albumin (BSA, 50 mg/mL) in a capped 5-mL vial equipped with a magnetic stir-bar and incubated at 37 °C. The final concentration of copolymer was 1.0 mg/mL. After 12 h and 36 h incubation, aliquots were withdrawn, diluted in 10-mM phosphate-buffered saline (pH 7.4 and 150 mM NaCl), and the solution turbidity of each sample was measured by UV-Vis absorption spectrometry in transmission mode. PtII-Release Assay from PtII-PNPs. Cumulative PtII-release amounts have been monitored at 37 °C in 10-mM phosphate buffer (pH 7.4) or 10 mM acetate buffer (pH 4.0), containing 150 mM NaCl. To this end, each sample solution of PtII-PNPs (2.5 mL, 6.4 mM in terms of PtII concentration) was dialyzed, using Slide-A-LyzerTM Dialysis Cassette G2 (3.5 kDa MWCO, 3 mL), against the corresponding buffer solution (500 mL) at 37 °C with vigorous stirring. An aliquot (10 mL) of the buffer solution outside the dialysis membrane was collected at a


37


Page 38 of 45

predetermined time and the concentration of PtII was monitored with SnCl2-mediated colorimetric analysis. Statistics. Statistical analysis was performed with Origin. All data were expressed as mean ± SD.

Statistical differences between groups in BSA study were assessed by the one-way

ANOVA, followed by Tukey’s post-hoc analysis. P < 0.05 was considered as the statistical significance level.

ACKNOWLEDGMENT This research was supported by Basic Science Research Program through the National Research Foundation

of

Korea

(NRF)

funded

by

the

Ministry

of

Education

(NRF-

2016R1D1A1B03934733) and the 2018 Research Fund of the Catholic University of Korea.

Supporting Information. Polymerization of block-copolymer excipients, 1H NMR spectra of copolymers and of PtII-PNPs, additional aqueous-phase SEC chromatograms of copolymers and PtII-PNPs showing narrow dispersity (Đ), SEC calibration curve, agitation time-dependent apparent mass change of PtII-PNPs (4k-g-15k), zeta (ζ) potential and DLS size distribution of PtII-PNPs in salt-free and high-salt conditions, core diameter (Dc) measurements of PtII-PNPs in TEM images, determination of cmc using pyrene and Nile red, DH changes of PtII-PNPs after lyophilization/rehydration, and SnII-mediated colorimetric quantification of PtII ions.

This

material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author Address correspondence to: [email protected]

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SYNOPSIS TOC


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Cisplatin-Encapsulated Polymeric Nanoparticles with Molecular

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