Letter pubs.acs.org/macroletters
Compact Vesicles Self-Assembled from Binary Graft Copolymers with High Hydrophilic Fraction for Potential Drug/Protein Delivery Yupeng Wang,†,‡ Lina Wang,†,§ Bin Li,*,∥ Yanxiang Cheng,† Dongfang Zhou,*,† Xuesi Chen,† Xiabin Jing,† and Yubin Huang*,† †
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § School of Materials Science and Engineering, Tianjin University, Tianjin, 300072, People’s Republic of China ∥ Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States S Supporting Information *
ABSTRACT: Hollow vesicles self-assembled from amphiphilic copolymers are of great interest in biomedicine field as drug and protein carriers. Efficient preparation of polymeric vesicles with high stability in vivo is highly desirable. Herein, a novel cooperative self-assembly of two graft copolymers (GCPs) with reversed hydrophilic−hydrophobic segments is investigated to achieve morphology control for biomedical application. Interestingly, nanosized vesicles are obtained for the binary system with relatively high hydrophilic fraction ( f hydrophilic, ∼60%), contrary to what is found in its singlecomponent counterpart. The cooperative self-assembly endowed the hybrid vesicles with excellent resistance to protein adsorption, prolonged blood circulation time, as well as low leakage of hydrophilic drugs/proteins. Furthermore, the biological activity of the protein is well preserved inside the cooperative vesicles, making it a promising candidate as the protein carrier.
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structure, with two fatty acid tails and a phosphate-linked headgroup. Together with other types of lipids, it assembles into the lipid bilayer which serves as the structural scaffold for other components.17,18 In this aspect, the hybrid assembly of nonlinear polymer structures can serve as a bioinspired polymeric self-assembly route.12,19,20 As one of the unique molecular architectures, graft copolymer (GCP) shows the advantages in self-assembled aggregates with multiple morphologies and structures.21−23 Especially, many researches have demonstrated that the vesicles formed by GCPs displayed enhanced resistance to nonspecific protein adsorption and controlled drug release behavior through tuning the hydrophilic−hydrophobic balance.24,25 However, almost no attention has been devoted to the cooperative self-assembly studies of binary GCPs, partly due to their relatively challenging synthesis and characterization.23,26 Thus, the understanding of structure− function relationship of binary GCP self-assembly systems is urgently needed, and also of practical importance in design of versatile carriers for biomedical application. Herein, we investigate, for the first time, the cooperative selfassembly of binary GCPs with reversed hydrophilic−hydro-
ver the last two decades, amphiphilic block copolymers (BCP) self-assembled vesicles, or polymersomes (PMs), have attracted considerable attentions in view of their potential application in biotechnology and materials science.1,2 It is well established that PMs exhibit enhanced structural stability and diverse cargo loading compared to liposomes and polymeric micelles.3−5 Further molecular designs of the polymer structure and functionality have solidified polymeric vesicles as versatile vectors in biomedicine, microreactor, and synthetic protocell.6−8 However, problems still exist about the basic issues as an ideal carrier compared with its biological counterpart, such as antifouling property against plasma proteins, long blood circulation time, low membrane permeability to loaded agents, and so on.5,9,10 As an alternative method to new polymer design, the cooperative self-assembly of two or more components can offer a facile but generic route to regulate the self-assembly morphology.11−13 Recently, Jiang and co-workers had fabricated asymmetric vesicles with different corona thickness through a cooperative self-assembly of binary BCPs (PS-b-PAA and PS-b-P4VP) mixture.14 As we know, the molecular architecture of copolymers is an important factor to control the morphology and surface functionality of the assemblies.15,16 On the other hand, the most abundant lipid found in cell membranes is the phospholipid, and it shows a nonlinear © XXXX American Chemical Society
Received: July 27, 2017 Accepted: October 9, 2017
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DOI: 10.1021/acsmacrolett.7b00549 ACS Macro Lett. 2017, 6, 1186−1190
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ACS Macro Letters phobic segments. Of interest, the two GCP structures we selected, poly(caprolactone)-g-poly(ethylene glycol) (PCL-gPEG, GCPo‑i) and poly(ethylene glycol)-g-poly(ε-caprolactone) (PEG-g-PCL, GCPi‑o), had the same composition of hydrophilic/hydrophobic chains, which formed the perfect pair as a binary system. By adjusting the fraction of hydrophilic segment (f hydrophilic), the binary GCPs could assemble into diverse morphologies in aqueous solution, which included a vesicle structure at f hydrophilic as high as 60%. The structure characteristics of the hybrid vesicle were further studied systematically as the drug/protein carrier, including the ability to resist protein adsorption, in vivo stability, cargo leakage at physiological pH, and the retention of protein bioactivity (Scheme 1). Scheme 1. Schematic Illustration of the Compact Vesicle Formation from Binary Graft Copolymers with High Hydrophilic Fraction for Potential Drug/Protein Delivery
Figure 1. Self-assembled morphology of PCL-g-PEG, PEG-g-PCL and 1:1 mixture (weight ratio) observed by TEM. (A) GCPo‑i30 and (B) GCPi‑o30 with f hydrophilic = 30% tended to form the vesicle structure. While with higher f hydrophilic (45% and 60%) all the single graft copolymers favored the micellar structure, such as (C) GCPi‑o60. The binary graft copolymer systems, (D) GCPmix30, (E) GCPmix45 and (F) GCPmix60, formed the irregular micelles, incompact petaling micelles, and vesicles, respectively.
the binary mixtures of GCPo‑i and GCPi‑o showed different selfassembly behaviors. By blending the GCPo‑i and GCPi‑o with the same f hydrophilic, it was found that the GCPmix30 formed irregular micelles, replacing their original vesicles morphology (Figure 1D), while GCPmix45 tended to form incompact petaling micelles (Figure 1E). Interestingly, as the f hydrophilic of binary GCPs increased up to 60% (GCPmix60), hollow vesicles (PMmix60) with diameter of 170 nm were observed (Figure 1F). A perusal of the literature showed all the single components of BCPs and GCPs were found to form vesicles at f hydrophilic < 50%, especially within the range of 25−45%, and the vesicles formation at such a high f hydrophilic was exceptional.23,24,27−29 It implied the distinctive packing pattern of binary GCPs during the cooperative self-assembly, which demands the detailed molecular understanding of nanostructure evolution. Here, some structural features of the hybrid vesicle will be discussed afterward when it is used as the drug/protein carrier. The high f hydrophilic of formed vesicles apparently increased the proportion of PEG coverage, which would reduce the capture by the reticuloendothelial system (RES) and prolong the circulation lifetime.30−32 Herein, nonspecific protein adsorption studies were first performed with the three type of PMs (PMo‑i30, PMi‑o30, and PMmix60) against FITC-labeled bovine serum albumin (FITC-BSA). ζ-potential of the PMs was close to zero, avoiding the static adsorption of proteins (Table S2). After incubation with FITC-BSA solution (PBS 7.4, 30 g/ L) for 48 h, there was a detectable increase in particle size for both of PMo‑i30 and PMi‑o30. In contrast, negligible change was observed for PMmix60, indicating a more obvious steric repulsion effect against protein adsorption and particle aggregation owing to the higher proportion of PEG segment (Figure 2A).33−35 By quantifying the nonadsorbed FITC-BSA via UV−vis spectroscopy, it was found that 4.25 ± 1.34% of FITC-BSA was absorbed onto the PMmix60, significantly lower than those of PMo‑i30 (8.78 ± 1.47%) and PMi‑o30 (11.68 ± 2.23%; Figures 2B and S8A). Similar results were also found for the protein adsorption on the corresponding polymeric films (Figure S8B,C). In vivo blood clearance study was further performed
First, the two types of GCPs, PCL-g-PEG (GCPo‑i) and PEG-g-PCL (GCPi‑o), were synthesized with different f hydrophilic (30%, 45%, and 60%, respectively; Tables 1 and S1, Schemes Table 1. Characterization of the Structure and Self Assembled Behavior of GCPs sizeb (nm)
f hydrophilica
copolymer
compositiona
CAC (mg/L)
30%
GCPo‑i30 GCPi‑o30 GCPmix30
PCL-g-PEG PEG-g-PCL 1:1d
1.02 0.95 1.95
123 142 140
GCPo‑i45 GCPi‑o45 GCPmix45
PCL-g-PEG PEG-g-PCL 1:1
1.90 1.49 2.85
140.7 147.7 155.1
GCPo‑i60 GCPi‑o60 GCPmix60
PCL-g-PEG PEG-g-PCL 1:1
2.14 5.87 3.63
181.4 115.8 169.6
45%
60%
morphologyc vesicle irregular micelle micelle petaling micelle micelle vesicle
a
Calculated by 1H NMR. bDetermined by DLS. cObserved by TEM. d Ratio in weight.
S1 and S2, and Figures S1−S4). Due to the amphiphilic nature, the GCPo‑i and GCPi‑o, and their binary mixtures (GCPmix) could self-assemble into nanoaggregates in selective solvent (water) with relatively low critical aggregation concentration (CAC) values (10−3 g/L), which ensured the stability of the nanoaggregates to a certain extent under the dilution condition (Figure S5). The self-assembly morphologies and particle sizes of these nanoaggregates were observed by TEM and DLS (Figures 1, S6, and S7). All the GCPo‑i and GCPi‑o exhibited the regular self-assembly behaviors, in which low f hydrophilic (30%) resulted in vesicles (PMo‑i30 and PMi‑o30; Figure 1A,B), and higher f hydrophilic (45% and 60%) favored the formation of regular micelles (Figure 1C and S7). Beyond our expectation, 1187
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Scheme 2. Schematic Representation of the Structure of PMsa
a
PMmix60 with two different stacking patterns displays more compact structure, benefit for the least adsorption of protein, and leakage of encapsulated agents.
Figure 2. Comparison of nonspecific protein adsorption and structural stability among the three types of polymersomes (PMs). (A) Change of particle sizes and (B) quantification of protein adsorption in three types of PMs in the presence of FITC labeled bovine serum albumin solution (FITC-BSA, 30 g/L) at 37 °C for 48 h. **P < 0.01. (C) Blood clearance curves of the three types of PMs in mice after in vivo injection [data are expressed as percent injected dose (%ID) ± standard deviation, n = 3, *P < 0.1]. (D) In vitro Dox release profiles of three types of polymersome-encapsulated Dox (PM@Dox) in PBS 7.4 at 37 °C for 72 h. **P < 0.01.
protein in blood which is responsible for gas transmission for the whole body, was chosen as a model protein and encapsulated into the PMs. As anticipated, the spherical morphology of PM@Hb was well preserved after Hb encapsulation, with a relatively larger particle size detected by DLS (Figures 3A, S10, and S11A and Table S4). SDS-PAGE
given its excellent antifouling capability. As shown in Figure 2C, the circulating half-life (T1/2) of rhodamine B labeled PMmix60 (RhB-PMmix60) was about 6.80 ± 0.31 h, longer than those of RhB-PMo‑i30 (5.49 ± 0.29 h) and RhB-PMi‑o30 (5.18 ± 0.54 h), confirming the slow clearance rate of PMmix60 from blood. All these results together offered compelling evidence that the high f hydrophilic of PMmix60 was superior to resist protein adsorption which could further enhance the blood residence time. Meanwhile, as potential carriers for therapeutic agents, the structural stability of PMs was important to prevent the leakage of cargos in bloodstream which may cause the off-target toxicity. Here, doxorubicin·HCl (Dox), a small-molecule anticancer drug, was encapsulated in the three types of PMs (Figure S9 and Table S3), and the drug release profiles were monitored by incubation of the resultant PM@Dox in physiological buffer (PBS 7.4). As shown in Figure 2D, only 29.7% of Dox was released from the PMmix60@Dox after 72 h, which was a much lower release rate compared with the singlecomponent PM@Dox (PMo‑i30@Dox: 42.1%, PMi‑o30@Dox: 46.8%). The different drug release profiles of these PMs may be attributed to their particular assembly structures. As proposed in Scheme 2, in the PMs, the insoluble PCL segments constitute the vesicle wall. PMo‑i30 involves the looping of hydrophobic PCL backbone into the core and the tailing of grafted hydrophilic PEG to form the corona.24 On the contrary, PMi‑o30 involves the tailing of grafted hydrophobic PCL into the core and the looping of hydrophilic PEG backbone to form corona.23 Specially, the binary PMmix60 combined the two different stacking patterns of the single-component PMs to form more compact hydrophobic and hydrophilic layers without gap (cross-linking-like structure), resulting in the least adsorption of protein and leakage of encapsulated agents. In addition to transport small molecular drugs, vesicles are also commonly used for protein delivery.36−38 As an ideal protein carrier, it was essential to maintain the integrity, bioactivity, and low leakage of proteins after encapsulation. Herein, hemoglobin (Hb), the most abundant functional
Figure 3. Characterization of PMmix60@Hb. (A) TEM morphology of PMmix60@Hb. The PMs transformed from hollow spheres to solid spheres after the internalization of free Hb. (B) In vitro Hb release profiles of the three types of polymersomes-encapsulated hemoglobin (PM@Hb) in PBS 7.4 at 37 °C for 72 h. **P < 0.01. (C) UV−vis spectra of PMmix60@Hb at different gas-binding states. (D) Oxygen dissociation curves of PMmix60@Hb (P50 = 29.5 mmHg) and native Hb (P50 = 27 mmHg).
results confirmed that Hb was entrapped within the cavity of the PM rather than absorbed on the surface (Figure S11B). Similar to the release profiles of Dox, a much lower protein leakage (∼10%) was found for PMmix60@Hb within 72 h, compared with those of PMo‑i30@Hb (17%) and PMi‑o30@Hb (25%), which will potentially reduce the renal toxicity caused by free Hb leaked into the bloodstream (Figure 3B). The encapsulated Hb also retained the capacity to combine and release oxygen. UV−vis spectra of PMmix60@Hb in different gasbinding states (CO, O2, N2) showed no change compared with free Hb except for a baseline drift caused by the scattering effect of the nanosized PMs (Figure 3C and S11C). Meanwhile, the oxygen affinity of PMmix60@Hb (P50 = 29.5 mmHg, Hill coefficient = 2.507) was similar to the native Hb (P50 = 27 mmHg, Hill coefficient = 2.254) (Figures 3D and S11D). 1188
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Charged proteins such as electronegative bovine serum albumin (BSA) and electropositive lysozyme (LYZ) could be also encapsulated in PMmix60 (Figure S12 and Table S5). Moreover, the PMmix60@Hb possessed excellent biocompatibility and blood compatibility even at high concentrations (Figures S13 and S14). All these results revealed that the PMmix60 can serve as a promising delivery carrier for proteins with preserved bioactivity and low leakage in physiological condition. In summary, we reported a compact vesicle structure with fraction of hydrophilic segment as high as 60% based on the cooperative self-assembly of binary graft copolymers with reversed hydrophilic−hydrophobic segments. Compared with the regular vesicles assembled by single-component graft copolymers with low fraction of hydrophilic segment (30%), the binary vesicle (PMmix60) showed superior resistance to protein adsorption, prolonged blood circulation time and lower leakage of hydrophilic drugs/proteins. Meanwhile, as the potential universal protein carrier, PMmix60 effectively preserved the original biological activity of the protein. Importantly, the cooperative self-assembly of multiple polymer architectures provides a new method to tune the self-assembly morphologies and functions, which encourages an intense investigation in this field.
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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/acsmacrolett.7b00549. Synthesis procedure and additional spectroscopies (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Bin Li: 0000-0003-1411-8731 Xuesi Chen: 0000-0003-3542-9256 Yubin Huang: 0000-0002-5212-318X Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 51403198, 51673188, and 51773198) and Jilin Provincial Science and Technology Department (Nos. 20150520019JH and 20170101091JC).
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ABBREVIATIONS GCP, graft copolymers; BCP, block copolymers; PMs, polymersomes; GCPo‑i, PCL-g-PEG, graft copolymers with the hydrophobic backbone and hydrophilic side chains; GCPi‑o, PEG-g-PCL, graft copolymers with the hydrophilic backbone and hydrophobic side chains; GCPmix, binary mixtures of the two types of graft copolymers 1189
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