Biomimetic star-shaped poly(ε-caprolactone)-b-glycopolymer block copolymers with porphyrin-core for targeted photodynamic therapy

Abstract

In this study, star-shaped porphyrin-cored poly(ε-caprolactone)-b-poly(gluconamidoethyl methacrylate) block copolymers (SPPCL-PGAMA) were successfully obtained. The synthetic route was via the ring-opening polymerization (ROP) of ε-caprolactone using a tetra-hydroxyethyl terminated porphyrin as a core initiator followed by the atom transfer radical polymerization (ATRP) of unprotected gluconamidoethyl methacrylate (GAMA) in 1-methyl-2-pyrrolidinone (NMP) solution at room temperature. The structure of the copolymer was thoroughly studied by nuclear magnetic resonance spectroscopy (NMR), Gel Permeation Chromatography (GPC), Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Notably, the as-prepared SPPCL-b-PGAMA that formed different structures being used for drug delivery systems has been researched. Moreover, this copolymer can release singlet oxygen under light irradiation and the singlet oxygen could be used for photodynamic therapy. In particular, UV-vis analysis showed that SPPCL-b-PGAMA has a very specific recognition with Concanavalin A (Con A) which provides porphyrin-cored SPPCL-b-PGAMA block copolymers for targeted drug delivery.

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Introduction

Porphyrin used for designing and synthesizing new functionalized materials has attracted a great deal of attention in very recent years.^1,2 However, drawbacks like hydrophobicity and non-selectivity result in molecular complexity, self-quenching, and photo-toxicity to the skin.^3,4 In very recent years, studies on biomimetic star-shaped polymer–porphyrin conjugates have been steadily increasing which could solve the aforementioned drawbacks.⁵ Frechet^6,7 and our group⁸ reported a functional porphyrin-cored polymeric shell based on Ring Opening Polymerization (ROP) of ε-caprolactone (CL) or L-lactic (L-LA). Holder⁹ and Cornelissen¹⁰ synthesized a series of star polymers with porphyrin core using Atom Transfer Radical Polymerization (ATRP). However, problems like poor water solubility and difficulties in synthesis process greatly limited its further application. Considering these problems, a hydrophilic polymer shell like PEG coupled with PCL-porphyrin for chemotherapy/PDT using,⁴ or PH-sensitive block copolymer POEGMA-PCL-porphyrin¹¹ were well synthesized.

Synthetic glycopolymers polymers have no carbohydrate backbone but own carbohydrate, which have been used to elucidate the specific sugar–protein recognition processes in living cells that hold great relevance for both drug discovering and bio-materials applications. Glycosylation could provide a possibility to specific interact with lectin type receptors that overexpressed in a specific malignant tissue.^12,13 Until now, limited research was done on the synthesis of glycopolymers–porphyrin with well control over both the molecular weight and architecture.^13–15 However, Dong provided a serials of hydrophilic chains composed of some glycopolymers, render the micelles enable to not only improved water solubility and micelle stability, but also provided a specific sugar–protein recognition processes.¹⁵ As possible as we can, no report was found out on the synthesis of amphiphilic star-shaped porphyrin-conjugated-glycopolymer block copolymers. In these work, it is very meaningful to use the glycopolymers with porphyrin core as recognition motifs. Moreover, it has been known that naturally oligosaccharides/polysaccharides plays an important function to stabilize the molecular structure of protein and then markedly improve the compatibility between hydrophobic polymers and hydrophilic peptide drugs.^16,17 Hydrophobic chemotherapeutic agents such as paclitaxel can then be encapsulated into the core region of this micelle.

Specifically, we synthesized a new class of biodegradable and biomimetic star-shaped porphyrin-cored poly(ε-caprolactone)-b-poly(gluconamidoethyl methacrylate) (SPPCL-b-PGAMA) block copolymers via direct ATRP of unprotected D-gluconamidoethyl methacrylate glycomonomer (GAMA), as shown in Scheme 1. The molecular structures, self-assembly and recognition properties of as-synthesized SPPCL-b-PGAMA have been thoroughly characterized by means of ¹H NMR, FT-IR, GPC, UV-vis, fluorescent spectroscopy, and TEM. In addition, its potential as a drug delivery carrier was also evaluated. Consequently, this will not only provide a specific targeting glycomonomer-PCL-based block copolymer but also provide potentially porphyrin-cored star-shaped SPPCL-b-PGAMA copolymers for combined chemotherapy/PDT strategy in one delivery system.

Scheme 1 Synthesis of star-shaped porphyrin-cored SPPCL-b-PGAMA block copolymers.

Experiments

Materials

Stannous octoate (SnOct₂), bipyridine, copper(I) bromide, 2-bromoisobutyryl bromide, Concanavalin A (Con A), 2-aminoethyl methacrylate hydrochloride and D-gluconolactone were purchased from Aldrich and these agents were used without further purification. Both ε-caprolactone (CL, Aldrich) and toluene were distilled from CaH₂. Paclitaxel was purchased from Jiangsu Yew Pharmaceutical Co., Ltd used as received. SPPCL was synthesized according to our previous publication (94.5% yield).^{8 1}H NMR (CDCl₃) of SPPCL₁₅ sample: δ (ppm) = 1.35–1.45 (m, 30H, –COCH₂CH₂CH₂CH₂CH₂O–), 1.55–1.79 (m, 60H, –COCH₂CH₂CH₂CHCH₂O), 2.28–2.43 (m, 30H, –COCH₂CH₂CH₂CH₂CH₂O–), 3.65 (t, 2H, –CH₂OH), 4.02–4.19 (m, 30H, –COCH₂CH₂CH₂CH₂CH₂O–). D-Gluconamidoethyl methacrylate glycomonomer (GAMA) was synthesized from D-gluconolactone and 2-aminoethyl methacrylate hydrochloride according to the literature procedure¹⁵ (52.0% yield), and ¹H NMR (D₂O): δ (ppm) = 1.75 (s, 3H, –CH₃), 3.38–3.70 (m, 6H, –CH₂NH– and –CHOH–CHOH–CH₂OH), 3.91–3.95 (t, 1H, –CHOH–), 4.13–4.19 (m, 3H, –OCH₂– and –COCHOH–), 5.58 (s, 1H, =CH₂), 6.0 (s, 1H, =CH₂). All other reagents and solvents were used without further purification.

Instrumentation

Nuclear magnetic resonance spectroscopy (NMR) spectra were recorded at room temperature on a Varian Mercury-400 spectrometer. Gel Permeation Chromatography (GPC) were determined on Perkin-Elmer Series 200 with a refractive index detector at 30 °C, and the elution phase was DMF (0.01 mol L⁻¹ LiBr) (elution rate: 1.0 mL min⁻¹) which used polystyrene as the calibration standard. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet FT-IR spectrophotometer (Nexus 470, Thermo Electron Corporation) at frequencies ranging from 400 to 4000 cm⁻¹. The differential scanning calorimetry (DSC) analysis was carried out using a Perkin-Elmer Pyris 1 instrument under nitrogen flow (10 mL min⁻¹). All samples were heated from 0 °C to 200 °C at 10 °C min⁻¹. Fluorescent spectra were performed at room temperature using a luminescence spectrometer (Cary Eclipse, AUS). Transmission electron microscope (TEM) micrographs were taken with a JEOL-JEM-2010 (JEOL, Japan) operated at 200 kV. One drop of aggregates solution was deposited onto the surface of 300 mesh Formvar-carbon film-coated copper grids. Excess solution was quickly wicked away with a filter paper. The image contrast was enhanced by negative staining with phosphotungstic acid (0.5 wt%).

Preparation of star-shaped tetra(2-bromo-2-methylpropionyl)-terminated poly(ε-caprolactone) with porphyrin-core (SPPCL-Br)

A typical example is given below. Firstly, SPPCL (150 mg, 0.0127 mmol) was dissolved in distilled CH₂Cl₂ under stirring followed by added triethylamine (351 mg, 3.47 mmol) under argon at room temperature. Then, the temperature of the mixture was decreased to 0 °C by ice bath. Finally, the solution of 2-bromoiso-butyryl bromide (789 mg, 3.47 mmol) in distilled CH₂Cl₂ (20 mL) was added dropwise into the mixture within 30 minute. After reacted for 24 hours at room temperature, the reacted mixture solution was washed with NaHCO₃ aqueous solution (100 mL) and deionized water (100 mL × 2). The organic phase was dried overnight with MgSO₄ followed by evaporated the solvent and the obtained product was purified by cold methanol. ¹H NMR (CDCl₃) of SPPCL-Br sample: d (ppm) = 9.16 (s, 8H, b-pyrrole-H), 8.18 (d, 8H, m-Ar-H), 7.35 (d, 8H, o-Ar-H), 1.32–1.49 (m, 30H, –COCH₂CH₂CH₂CH₂CH₂O–), 1.51–1.78 (m, 60H, –COCH₂CH₂CH₂CH₂CH₂O–), 1.93 (s, 6H, –C(CH₃)₂Br), 2.25–2.39 (m, 30H, –COCH₂CH₂CH₂CH₂CH₂O–), 4.01–4.14 (m, 30H, –COCH₂CH₂CH₂CH₂CH₂O–).

Synthesis of biomimetic star-shaped porphyrin-cored SPPCL-b-PGAMA block copolymers

A typical procedure for the ATRP of unprotected GAMA glycomonomer using SPPCL-Br macroinitiator is as follows: firstly, both SPPCL-Br (0.002 mmol, 28.92 mg) and GAMA glycomonomer (0.59 mmol, 181.7 mg) were dissolved in NMP (0.5 mL) at room temperature, and the mixture solution was degassed via nitrogen purge for 30 min. Then, copper(I) bromide (0.008 mmol, 1.2 mg) and bipyridine (0.0016 mmol, 2.5 mg) were added in turn, and stirred vigorously under nitrogen at room temperature for 24 hours. Finally, the reacted mixture was precipitated into isopropanol (30 mL), and then purified sequentially by tetrahydrofuran (2 mL) and heated methanol (5 mL) to remove the possible unreacted SPPCL-Br macroinitiator and GAMA glycomonomer. The resulting block copolymer was then dried in vacuo overnight at 40 °C (81.8 mg, 71.4% yield).

Preparation of glucose-installed aggregates in water

The solution of SPPCL-b-PGAMA copolymer dissolved in DMF at room temperature was added distilled water dropwise with a microsyringe. After stirring for 24 hours, DMF was removed by a dialyzing method. The morphology of aggregates was determined by TEM.

Measurement of the critical micelle concentration (CMC) of SPPCL-b-PGAMA

The CMC of amphiphilic SPPCL-b-PGAMA copolymer in aqueous solution was studied on a luminescence spectrometer and pyrene was used as a hydrophobic fluorescent probe.¹⁸ 100 μL of pyrene solutions (6 × 10⁻⁶ M in methanol) were added to containers (5 mL), and the methanol was allowed to evaporate. Copolymer aqueous solutions at different concentrations were then added to the containers containing the pyrene residue. Each sample with various polymer concentrations from 0.00005 to 1 mg L⁻¹ contained the same concentration of pyrene residue. Emission wavelength was carried out at 688 nm, and excitation spectra were recorded, ranging from 300 to 360 nm. The excitation and emission bandwidths were 5 and 5 nm, respectively. From the pyrene excitation spectra, the intensity ratio at 344 nm was analyzed as a function of the polymer concentration.

Detecting singlet oxygen (¹O₂) production

In our study, chemical method using 1,3-diphenylisobenzofuran (DPBF) was proposed for detecting ¹O₂ generated by SPPCL-b-PGAMA photosensitizers (TPPH₂ as a standard regent),^4,19,20 DPBF reacts irreversibly with ¹O₂ that causes a decrease in the intensity of the DPBF absorption band at 456 nm. In a typical experiment, 30 μL TPPH2 (1.5 × 10⁻⁵ mol L⁻¹), SPPCL (1.5 × 10⁻⁵ mol L⁻¹) or SPPCL-b-PGAMA (1.5 × 10⁻⁵ mol L⁻¹) was mixed with 3 mL of DPBF (10⁻⁶ mol L⁻¹) in DMF respectively. Then, the solutions were irradiated with a 650 nm laser source (5 mW) and their absorbencies at 456 nm were recorded every 30 seconds in a luminescence spectrometer.

Lectin recognition^12,13,21

The lectin recognition activity of the copolymer solution was analyzed by changes in the turbidity of solution with time at 360 nm following the addition of various concentrations of aggregate solution into Con A solution, in which the concentration of Con A was equal to 0.5 mg mL⁻¹.

Preparation of paclitaxel-loaded nanoparticles in aqueous solution

Using a dialysis method, typical procedure for the fabrication of paclitaxel-loaded nanoparticles in aqueous solution is as follows.^22,23 SPPCL-b-PGAMA (8 mg) and paclitaxel (2 mg) were dissolved in 2 mL of DMF. Distilled water (8 mL) was then added gradually using a microsyringe until the formation of nanoparticles. The obtained nanoparticles solution was then put into a dialysis bag and subjected to dialysis against 4 × 1 L of distilled water for 24 h. The drug-loaded nanoparticles solution was lyophilized and stored at 4 °C. The drug-loaded nanoparticles (2 mg) were dissolved in 2 mL of acetonitrile and the loading efficiency of paclitaxel in SPPCL-b-PGAMA was determined by HPLC.

Loading efficiency (L.E.) (%) = (weight of paclitaxel in the SPPCL-b-PGAMA/weight of the feeding paclitaxel) × 100%

Encapsulation efficiency (E.E.) (%) = (weight of paclitaxel in the SPPCL-b-PGAMA/weight of the drug-loaded micelles) × 100%.

In vitro paclitaxel release from drug-loaded nanoparticles²⁴

The drug-loaded nanoparticles (4 mg) and free paclitaxel were directly immersed into 4 mL of buffer solution (0.01 M, pH = 7.4, 0.1% sodium salicylate (w/v)) and then the mixture solution was put into a dialysis membrane bag (MWCO = 7000 Da). Then, it was put into a container against 26 mL buffer solution at 37 °C. The drug-released solution was extracted 5 mL periodically and was replaced with an equal volume of fresh buffer solution. The amount of the paclitaxel released from nanoparticles was determined by the HPLC system.

Results and discussion

Synthesis of biomimetic star-shaped SPPCL-b-PGAMA block copolymers

Currently, well-defined glycopolymer has been synthesized by controlled polymerization techniques such as ring-opening metathesis polymerization, nitroxide-mediated radical polymerization, Atom Transfer Radical Polymerization (ATRP), and reversible addition–fragmentation chain transfer polymerization (RAFT). Particularly, ATRP technique proved to be a robust strategy for the preparation of well-defined glycopolymer since the direct polymerization of unprotected glycomonomer provides a very convenient method to generate glycopolymer. In this work, biomimetic star-shaped SPPCL-b-PGAMA block copolymers were synthesized by the method of direct ATRP of unprotected GAMA in polar NMP solvent via three steps shown in Scheme 1. Firstly, well-defined star-shaped PCL with four hydroxyl end groups (SPPCL) was synthesized by the controlled ring-opening polymerization of ε-caprolactone monomer according to our previous publications.⁸ Then, by means of terminal acylated reaction, the SPPCL precursor was transformed into the SPPCL-Br with 2-bromo-2-methylpropionate end groups. Comparing the ¹H NMR of the resulting SPPCL-Br with that of the SPPCL precursor, the proton signals at 3.65 ppm assignable to the primary hydroxy methylene end group (–CH₂OH, δH^e) of the SPPCL precursor wholly disappeared while new signals corresponding to methyl protons of 2-bromo-2-methylpropionate (–C(CH₃)₂Br, δH^f) appeared at 1.84 ppm for the obtained SPPCL-Br (Fig. 1). Moreover, the integral ratio of the proton signal on the 2-bromo-2-methylpropionate end group to the repeating methylene unit of SPPCL-Br was very close to the theoretical value (H^a/H^f = 48/6). These results show that the hydroxyl end groups of the SPPCL-OH precursor were quantitatively converted into 2-bromo-2-methylpropionate end groups within SPPCL-Br, and the SPPCL-Br with low molecular weight was obtained similarly. Finally, the direct ATRP of unprotected GAMA glycomonomer was performed in NMP solution at room temperature and the results are summarized in Table 1. Compared with SPPCL-Br precursor, Fig. 2 shows the typical GPC curves (DMF solvent) of as-synthesized SPPCL-b-PGAMA copolymers with different PGAMA block, notably, the unimodal elution peak of the purified block copolymer is apparently shifted toward a higher molecular weight region coupled with a narrow polydispersity in comparison with that of the original SPPCL-Br. In addition, the number-average molecular weight of the as synthesized polymers SPPCL-b-PGAMA determined by GPC (M_n,GPC) were in well control when reacted with different molecular weight of PGAMA (Fig. 2). Moreover, the polymer molecular weight determined by ¹H NMR (M_n,NMR) is reasonably consistent with the theoretical molecular weight of polymer (M_n,th) where M_n,th = [M]/[I] × M_monomer × yield + M_initiator. Furthermore, the actual copolymer molecular weight determined by ¹H NMR (M_n,NMR) of SPPCL₂₄-b-PGAMA₃₇ block copolymers increases linearly with the molar ratio of GAMA glycomonomer to SPPCL-Br macroinitiator ([GAMA]/[SPPCL-Br]), and the molecular weight distribution is reasonably narrow (Fig. 2). This indicates that the molecular weight of the SPPCL-b-PGAMA block copolymer can be accurately predicted by the molar ratio of GAMA glycomonomer to macroinitiator, which is a characteristic of “living”/controlled radical polymerization. As a note, M_n,GPC is apparently higher than M_n,th, which can be attributed to the different hydrodynamic volume of SPPCL polymers when polystyrene was used as a calibration standard for GPC measurement.

Fig. 1 ¹H NMR spectra of SPPCL (1) and SPPCL-Br (2) in CDCl₃.

Table 1 Synthesis of SPPCL-b-PGAMA copolymers via the ATRP of GAMA monomer in NMP solution at room temperature

Entry^a	[GAMA]/[SPCL-Br]	Monomer conv. (%)	fPCL/fPGAMA^b (%/%)	Mn,GPC^c	Mn,th^d	Mn,NM^e	Mw/Mn^c	Yield (%)
a The subscript numbers represent the repeating units of polymers.b fPCL/fPGAMA denotes the weight fractions of PCL and/or PGAMA within block copolymers, which was determined by ^1H NMR.c Mw/Mn of SPPCL-b-PGAMA denotes the molecular weight distribution of polymer, where weight-average molecular weight (Mw) and number-average molecular weight (Mn) are determined by GPC in DMF.d Mn,th = [GAMA]/[SPPCL-Br] × Mmonomer × yield + Minitiator, Mn,th denotes the theoretical number-average molecular weight of the SPPCL-b-PGAMA.e Mn,NMR of SPPCL-b-PGAMA was determined from the integral ratio of the signal on the main chain of PCL (–CH2–, 2.20–2.29 ppm) and the signal on the main chain of PGAMA (–CH2–, 1.67–2.00 ppm) from the ^1H NMR spectra.
SPPCL24-OH	—	80.0	100/0	21 133	11 407	11 812	1.42	96.3
SPPCL24-Br	—	—	100/0	23 514	13 768	14 146	1.56	84.0
SPPCL24-PGAMA37	133	73.3	21.0/79.0	50 792	52 244	57 248	1.72	73.3
SPPCL24-PGAMA18	36	70.2	35.0/65.0	29 654	33 816	33 918	1.61	75.2
SPPCL24-PGAMA3	12	53.2	79.0/21.0	24 343	15 296	15 498	1.46	62.4

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Fig. 2 GPC traces of as-synthesized SPPCL-b-PGAMA copolymers in DMF solution (1) and dependence of M_n,GPC and the molar ratio of [GAMA]/[SPPCL-Br] (2).

In the Fig. 3, Compared with the SPPCL-Br macroinitiator, the ¹H NMR (DMSO-d₆) of SPPCL-b-PGAMA₃₇ copolymers clearly shows that besides the typical proton signals of PCL backbone, new proton signals appeared at 1.67–2.00 ppm (δH^a) and 0.47–1.12 ppm (δH^b) for backbone of PGAMA glycopolymer, and at both 3.37–3.76 ppm and 4.36–4.95 ppm for the glucose residues, as shown in Fig. 3(1). Moreover, FI-IR Spectrum of the SPPCL-OH precursor, SPPCL-Br and the SPPCL-b-PGAMA copolymers were shown in Fig. S1.† Besides the distinct carbonyl stretching band at 1729 cm⁻¹ for PCL backbone, the SPPCL-PGAMA block copolymers presented the broad O–H band at 3396 cm⁻¹ assignable to the glucose residues of PGAMA block, and the characteristic amide I and II bonds at both 1645 cm⁻¹ and 1552 cm⁻¹ for the linker groups within PGAMA block were also appeared. Meanwhile, the relative intensities of carbonyl bands at 1729 cm⁻¹ within PCL block to the amide within PGAMA block decreased gradually with the increasing block length of PGAMA. Furthermore, the DSC analysis of SPPCL-PGAMA copolymers showed that the outer PGAMA blocks progressively hampered the crystallization of the inner PCL blocks, and the degree of crystallinity (X_c) of the PCL block within copolymers decreased dramatically from 66.1% to 0.5% with the increasing block length of PGAMA,²⁵ as shown in Fig. S2 and Table S1.† This suggests that the block copolymerization of bio-mimetic PGAMA glycopolymer with biodegradable PCL might greatly hamper the high crystallinity properties and the compatibility of hydrophobic PCL with hydrophilic peptide-based drugs for drug delivery. In all, the above results indicate that biomimetic star-shaped SPPCL-b-PGAMA block copolymers with different compositions were successfully synthesized via the direct ATRP of unprotected GAMA glycomonomer using SPPCL-Br macroinitiator in NMP solution at room temperature (Scheme 1).

Fig. 3 H¹ NMR spectrum of SPPCL-b-GAMA in the solution of deuterium DMSO (1) and D₂O (2).

Self-assembly behavior of the SPPCL-b-PGAMA

Fluorescence probe technique was used to study the micelle formation with a serial of different concentrations pyrene. It is known that the fluorescent properties of pyrene largely depend on its microenvironment. Once pyrene was preferentially partitioned from a hydrophilic into hydrophobic microenvironment, an abrupt increase in fluorescence intensity can be observed with increasing copolymer concentrations. As shown in Fig. S3,† the intensity of the pyrene excitation spectra versus the logarithm of the SPPCL-b-PGAMA copolymer concentrations was plotted. We determined the CMC by intersecting straight-line segments drawn through the points at the lowest polymer concentrations, lying on a nearly horizontal line, and through the points on the rapidly rising part of the plot according to previous study.²⁶ The CMC values of SPPCL-b-PGAMA₃, SPPCL-b-PGAMA₁₈ and SPPCL-b-PGAMA₃₇ were 0.00553, 0.0154 and 0.0372 mg mL⁻¹, respectively, suggesting that CMC increased with increasing hydrophilic segment under the same hydrophobic association. The result coincides with other experiment result and it helps to determine increasing concentrations for suitable temperature for pharmaceutical applications.

The morphology of the self-assembled aggregates from these SPPCL-PGAMA block copolymers was investigated by TEM, as shown in Fig. 4. To investigate the effect of PGAMA block length and/or the weight fraction (f) of PGAMA on the aggregates' morphology, the hydrophobic PCL block was kept at 24 repeating unit. When the hydrophilic PGAMA block was long (such as SPPCL₂₄-PGAMA₃₇, f_GAMA = 79%), the normally spherical micelles an average diameter of about 35 nm were given in Fig. 4(1). As the hydrophilic PGAMA block shortened (e.g., SPPLA₂₄-b-PGAMA₁₈, f_GAMA = 65%), it can be seen that worm-like aggregates were mainly presented. This phenomenon can be attributed to both the decreased repulsion among the corona chains (i.e. hydrophilic PGAMA corona) and the increased surface tension resulting from the increased hydrophobicity–hydrophilicity balance. Finally, with the increasing ratio of hydrophobicity to hydrophilicity, the morphological of the aggregates with an average diameter of about 320 nm were shown for SPPCL-b-PGAMA₃ sample [f_PGAMA = 21%, Fig. 4(3)]. These glucose-installed micelles were bigger than conventional polymeric micelles, and they probably do not have the simple core–shell micelle structure formed by conventional amphiphilic block copolymers. According to literature reports,¹⁵ larger complex micelles are often formed by the further aggregation of simple core–shell micelles, which is induced by hydrogen bonding and van der Waals interactions among the hydrophilic shells. Therefore, these larger glucose-in-stalled aggregates are possibly formed by inter-micellar aggregation of the simple micelles because of the strong hydrogen bond interactions among the glycopolymer shells. Furthermore the above results indicate that the glucose-in-stalled aggregates with different morphologies can be fabricated by adjusting the weight fraction of PGAMA block with in these copolymers, which is similar to the morphological transformation of polymeric aggregates reported for bio-degradable poly(L-lactide) – and/or PCL-b-poly(ethylene oxide) copolymers.²⁷ Significantly, this will provide a platform for studying the bio-molecular recognition between sugar and protein on nanoparticles surface because these sugar-installed aggregates might present different sugar densities and spatial distribution.

Fig. 4 TEM photographs of SPPCL-b-PGAMA copolymers: SPPCL₂₄-PGAMA₃₇ (1), SPPCL₂₄-PGAMA₁₈ (2) and SPPCL₂₄-PGAMA₃ (3).

UV-vis analyses

The obtained SPPCL-b-PGAMA copolymers were further characterized by UV-vis spectroscopy (Fig. S4†). As the same as porphyrin, the UV-vis spectra of SPPCL₂₄-b-PGAMA₃₇ showed the Soret (435 nm) and Q bands (500–700 nm), which are known to be the characteristic of porphyrin which indicates that the luminescent property of porphyrin moiety was retained within SPPCL₂₄-b-PGAMA₃₇. Thus, this will potentially enable SPPCL₂₄-b-PGAMA₃₇ used for the biological probe and photodynamic therapy applications.²⁸

Singlet oxygen (¹O₂) production of SPPCL-b-PGAMA plus irradiation

¹O₂ is the initial agent in the photodynamic therapy of cancer (PDT). The generation of singlet oxygen by photosensitizer was detected chemically using the DPBF as a detector. DPBF reacts irreversibly with ¹O₂, which causes a decrease in the intensity of the DPBF fluorescence absorption band at ∼400 nm. Fig. 5 shows the decrease in fluorescence intensity at 456 nm as a function of irradiation time. Moreover, in the case of free porphyrin TPPH₂, the fluorescence intensity of DPBF caused by TPPH₂ dropped sharply to 20 percent in two minutes which proves a large number of singlet oxygen produced during this period, however, the fluorescence intensity of DPBF declined slowly when mixed with SPPCL₂₄-b-PGAMA₃₇, in other words, the singlet oxygen production ability of SPPCL₂₄-b-PGAMA₃₇ can be well controlled by irradiation time, which indicates that this will be a promising technology for PDT.²⁹

Fig. 5 Fluorescence intensity (at 456 nm) decay curves of DPBF.

Recognition properties of star-shaped SPPCL-b-PGAMA block copolymers

In living systems, the specific sugar–protein recognition events govern many psychological and pathological processes, which hold great relevance for both drug discovery and biomaterials applications. It is reported that Con A specifically recognizes D-glucopyranoside and D-mannopyranoside residues with free 3-, 4-, and 6-hydroxyl groups, and the binding of Con A with glycopolymer usually results in the Con A-cross-linked aggregates. Thus, the interaction of Con A with the SPPCL₂₄-b-PGAMA₃₇ copolymer was investigated in aqueous solution at room temperature. The turbidity slightly increased with the copolymer concentration for SPPCL₂₄-b-PGAMA₃₇ sample, and no precipitation was observed in solution. This suggests that the binding between copolymer and Con A probably occurred and resulted in the Con A-cross-linked aggregates. This also suggests that the Con A-cross-linked aggregates were stable in aqueous solution and would not induce a dramatically change turbidity as measured by UV-vis (Fig. 6). In conclusion, the above analyses indicate that these SPPCL-b-PGAMA copolymers had specific binding with Con A in aqueous solution which show that this porphyrin-cored glycopolymer can be used for targeted drug delivery.

Fig. 6 The interactions of Con A (0.5 mg mL⁻¹) with SPPCL₂₄-PGAMA₃₇ at different concentration.

In vitro paclitaxel-release study

Pa Paclitaxel, an anticancer drug with a very low solubility in water, was used as a model drug to evaluate the prolonged release behavior of SPPCL₂₄-b-PGAMA₃₇ micelles. The paclitaxel loading efficiency (LE) of the star-shaped copolymer was about 12.7%, and encapsulation efficiency (EF) was 63.5%. This was probably attributed to both the interior cavity and the larger space within hydrophobic SPPCL core, suggesting that this star-shaped architecture of copolymer was suitable for the encapsulation of drug. Sodium salicylate, a hydrotropic agent, significantly increased the solubility of paclitaxel without destroying the micelle structure of SPPCL-b-PGAMA.^23,24,30 In Fig. 7, the two-phase-release profile was observed in both buffered solutions. In case of the paclitaxel-loaded nanoparticles of SPPCL-b-PGAMA (PMT), relatively rapid release in the first phase was observed after 12 hours, followed by a sustained and slower release over a prolonged period of time (69 h, 75.3%). In comparison with the release of free paclitaxel, the paclitaxel release from the micelle was notably much slower. In this case, Paclitaxel release can be anticipated after micelles are internalized in tumor cells via the endocytosis pathway. Consequently, enhanced bioavailability of the paclitaxel is essential for causing cancer cells death.

Fig. 7 Cumulative release curve of the free paclitaxel and the PMT in PBS.

Conclusion

Star-shaped porphyrin-cored SPPCL-b-PGAMA block copolymers were successfully synthesized from the direct ATRP of unprotected GAMA glycomonomer using SPPCL-Br in NMP solution at room temperature. When the hydrophilic PGAMA block increased, the morphology of the aggregates shows very different. Moreover, these SPPCL-b-PGAMA copolymers showed specific recognition with Con A because of stable Con A-cross-linked aggregates in aqueous solution. Furthermore, these block copolymers could self-assemble to form micelles acting as nanosized photosensitizing agents and further encapsulate hydrophobic drugs such as Paclitaxel.

Acknowledgements

The authors are greatly grateful for the financial support of the National Natural Science Foundation of China (21004031), the Natural Science Foundation of Jiangsu Province (BK2011459), the National Postdoctoral Foundation of China (20090461065), the National Postdoctoral Foundation of Jiangsu Province (1001034B). Open Foundation of Stake Key Laboratory of Natural and Biomimetic Drugs, Peking University (K20110105), and the social development Foundation of Zhen jiang (SH2012024).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07402e

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