A thermo- and pH-responsive poly(N-isopropylacrylamide)–Mn–ZnS nanocomposite for controlled release and real-time photoluminescence tracking of doxorubicin

Abstract

Smart drug carriers with intrinsic photoluminescence (PL) tracking of controllably released drugs are of great significance to indicate in real-time where, when and how the drug was released. Herein, we present a novel multifunctional poly(N-isopropylacrylamide)–Mn–ZnS (PMZS) nanocomposite for thermo- and pH-controlled release of doxorubicin (Dox) and real-time tracking of the released Dox in a PL-enhanced manner. The simple coupling of Mn–ZnS quantum dots and poly(N-isopropylacrylamide) resulted in the unique thermo- and pH-responsive PL of PMZS. There was a much higher PL intensity at 37 °C than 25 °C and a much higher PL intensity at weakly acidic pH than at neutral pH, which is promising for PL imaging in tumor microenvironments. The PL of the PMZS composite was greatly quenched upon loading of Dox (the loading content and encapsulation efficiency of PMZS composites toward Dox were 15.2% and 84.1%, respectively), but was gradually restored with the controllable release of Dox, and the restored PL intensity was proportional to the cumulative amount of Dox released. The versatility of the PMZS composite as an effective Dox carrier and real-time PL-enhanced tracking of the controllably released Dox were further proved by cell viability assays and in vitro cell imaging experiments. The simple PMZS nanocomposite was easily-synthesized, biocompatible, and promising for PL tracking of when, where and how Dox was released.

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Introduction

Biocompatible and smart drug carriers are widely explored for improving curative effects.¹ Numerous materials such as nanoparticle composites,^2–6 biodegradable polymers,^7,8 stimuli-responsive materials,^9,10 supramolecular switches^11,12 and liposomes,¹³ have been designed to store drugs in normal cells or tissues, and release drugs around tumors when triggered by temperature,¹⁴ pH,^7,15,16 enzymes,¹⁷ light,¹⁸ microwaves² and redox reactions.¹⁹ Therein, poly(N-isopropylacrylamide) (PNIPAm) has been very important for the design of thermo-responsive drug carriers for loading a drug at low temperature and releasing the drug at high temperature.^20–24 To endow PNIPAm-based drug carriers with other stimuli-responsive properties, other ingredients are usually incorporated, for instance, acrylic acid^25,26 and acrylamidophenyl boronic acid^27,28 have been incorporated for pH and glucose responsiveness, respectively.

Herein, we incorporated the famous Mn-doped ZnS quantum dots (QDs)^29–33 into PNIPAm for getting both the thermo- and pH-responsiveness and photoluminescence (PL) property by the simple one-pot synthesis procedure (Scheme 1). Mn-doped ZnS QDs have the special property of enhanced PL signals upon aggregation,^34–37 while PNIPAm shrinkage at high temperature exactly leads to the aggregation of Mn–ZnS QDs, consequently, the coupling of Mn-doped ZnS QDs and PNIPAm would definitely endow the poly(N-isopropylacrylamide)–Mn–ZnS (PMZS) nanocomposite the unique property of enhanced PL signals with the increase of temperature. Besides, the PL of Mn-doped ZnS QDs was pH-dependent, endowing the PMZS nanocomposite with the pH-responsiveness. Such intrinsic thermo- and pH-responsive PMZS nanocomposite would be suitable for the controlled drugs release and real-time PL tracking the released drugs. The latter was of great significance for indicating where, when and how the drugs were released.^38–42

Scheme 1 Procedure for synthesis of poly(N-isopropylacrylamide)–Mn–ZnS (PMZS) nanocomposite and PL-enhanced tracking of the controllably released Dox.

To prove that hypothesis, the famous doxorubicin (Dox) was chosen as the drug model. The PL of PMZS nanocomposite was quenched upon the loading of Dox at low temperature (e.g. <20 °C) and in neutral pH (pH 7.4), but restored upon the releasing of Dox at body temperature (37 °C) and in low acidic (pH 5.5–6.5) environment (Scheme 1). The restored PL intensity of PMZS nanocomposite could track the controllably released amounts of Dox in a noninvasive manner. Consequently, the easily-synthesized PMZS nanocomposite would be the special drug carrier, not only for the controlled release of Dox, but also for the real-time tracking where, when and how Dox was controllably released.

Experimental

Chemicals

All reagents were at least of analytical grade. N-Isopropylacrylamide (NIPAm), potassium persulfate (KPS), sodium dodecyl sulfonate (SDS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and Dox·HCl were from Aladdin (Shanghai, China). N,N-Methylene-bis-acrylamide (Bis) was from Amresco (OH, U.S.A.), ZnSO₄·7H₂O, Mn(CH₃COO)₂·4H₂O, and Na₂S·9H₂O were from Tianjin Kaitong Chemicals (Tianjin, China), the Second Chemicals (Shenyang, China), and Tianjin Sitong Chemicals (Tianjin, China), respectively. 2-(N-Morpholino)ethanesulfonic acid (MES) and tris(hydroxymethyl)methyl aminomethane (Tris) were from Guangfu Fine Chemical Research Institute (Tianjin, China). The MES–Tris buffer (10 mM, pH 5.0–7.4) were used for loading and releasing of Dox. All fresh solutions were prepared in ultrapure water (Wahaha, Hangzhou, China).

Apparatus

The transmission electron microscopy (TEM) on a JEM-2100F field emission transmission electron microscope (JEOL, Japan) was operated at a 200 kV accelerating voltage. The samples for TEM were obtained by freeze-drying sample droplets from water dispersion for 24 h onto a 300-mesh Cu grid coated with a lacey carbon film. The FT-IR spectra (4000–400 cm⁻¹) in KBr were recorded using a Magna-560 spectrometer (Nicolet, Madison, WI). The UV-vis absorption measurements were performed on a UV-3600 UV-vis-NIR Spectrophotometer (Shimadzu, Japan). The photoluminescence, lifetime and absolute quantum yield measurements were performed on a PTI QM/TM/NIR spectrometer (Birmingham, NJ, U.S.A.) equipped with 75 W xenon light and temperature controller. In vitro PL imaging of cells were acquired on Olympus reflected fluorescence system (Olympus BX53, Japan). MTT assays were acquired on a Synergy H4 Multi-Mode Microplate Reader (BioTek, U.S.A.).

Preparation of PMZS nanocomposites

Briefly, to a four-neck flask protected in argon atmosphere, NIPAm (1.77 mmol), SDS (1.00 mmol), Bis and ZnSO₄·7H₂O (amount as listed in Table 1) and ultrapure water (10 mL) were added and vigorously stirred. The mixture was heated to a certain temperature (25, 37, 50 and 70 °C) respectively, and then the aqueous solutions of TEMED (1 mL, 60 mM) and KPS (10 mL, 6 mM) were added slowly in sequence and the reactions were kept at the corresponding temperature for 4.5 h. The aqueous solution of Mn(CH₃COO)₂ (3 mL, amount as listed in Table 1) was injected into the flask once the temperature of the reactants was 30 °C, and then the mixture was stirred for 20 min, followed by the addition of 10 mL aqueous solution of Na₂S (amount as listed in Table 1). The mixture was stirred at 45 °C for 1.5 h, and then the mixture was aged under stirring at 35 °C overnight in air. Finally, the composite was dialyzed for 2 weeks to remove unbonded monomers and reagents.

Table 1 Synthesis conditions of PMZS nanocomposites

Entry	Bis (mmol)	ZnSO4·7H2O (mmol)	Mn(CH3COO)2·4H2O (mmol)	Na2S·9H2O (mmol)	Temperature (°C)
PMZS-1	0.022	0.56	0.060	0.86	25
PMZS-2	0.022	0.56	0.060	0.86	37
PMZS-3	0.022	0.56	0.060	0.86	50
PMZS-4	0.022	0.56	0.060	0.86	70
PMZS-5	0.022	0.48	0.051	0.73	37
PMZS-6	0.022	0.65	0.069	0.99	37
PMZS-7	0.022	0.78	0.078	1.20	37
PMZS-8	0.058	0.65	0.069	0.99	37
PMZS-9	0.124	0.65	0.069	0.99	37
PMZS-10	0.177	0.65	0.069	0.99	37

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The raw Mn–ZnS QDs were synthesized in parallel according to the same procedure but without the polymerization part. Typically, to a four-necked flask in argon atmosphere, 10 mL of ultrapure water, 0.56 mmol of ZnSO₄·7H₂O and 0.060 mmol of Mn(CH₃COO)₂·4H₂O were added and vigorously stirred for 20 min. Then 10 mL aqueous solution of 0.086 M Na₂S was injected into the solution. The mixture was stirred for 1.5 h at 45 °C in argon then aged at 35 °C overnight in air. Finally Mn–ZnS QDs were harvested via centrifuge, washed with ethanol for three times, and dried in a vacuum.

PL measurement

Typically PL was measured under the excitation of 320 nm, with slit widths of 2 nm and long-pass (400 nm) emission filter. For evaluation of thermo-responsibility, the solutions of PMZS (with equivalent Mn–ZnS of 0.015 g L⁻¹) in MES–Tris buffers (10 mM, pH 6.5) was equilibrated at certain temperature (22–42 °C) for 10 min before PL measurement. For test of pH-responsibility, 2 mL solution of PMZS-8 (0.06 g L⁻¹, with equivalent Mn–ZnS QDs of 0.015 g L⁻¹) or raw Mn–ZnS QDs (0.015 g L⁻¹) in MES–Tris buffer (10 mM, pH 5.0–7.4) was measured at 25 or 37 °C.

Loading and controllably releasing of Dox

For Dox loading, 0.15 mL aqueous solution of Dox·HCl (10 g L⁻¹) was mixed with 3 mL solution of PMZS nanocomposite (2.5 g L⁻¹, in 10 mM MES–Tris buffer, pH 7.4) by continuously stirring for 24 h at 20 °C. After that, the dispersion was centrifuged at 12 000 rpm for 7 min, and then the PMZS loaded with Dox (PMZS–Dox) was collected and washed for three times with cold ultrapure water to remove the free Dox. All the supernatants were merged and the amount of Dox in supernatants was determined by the absorbance at 478 nm (UV-vis spectra of Dox were given in Fig. S1 in ESI†). The Dox loading content was calculated as the weight ratio of Dox trapped in PMZS–Dox to PMZS–Dox, and the encapsulation efficiency was calculated as the weight ratio of Dox in PMZS–Dox to total Dox. The PL signals of 2 mL PMZS-8 (0.06 g L⁻¹) and PMZS-8–Dox (with equivalent PMZS-8 concentration of 0.06 g L⁻¹) in MES–Tris buffer (pH 7.4, 10 mM) was also measured to evaluate the PL change upon loading of Dox.

The release of Dox was performed in 5 mL centrifuge tube. PMZS-8–Dox (with equivalent PMZS-8 concentration of 2.5 g L⁻¹) in 3.0 mL of MES–Tris buffer (10 mM) with different pH (5.5, 6.5 and 7.4) was stirred at 25 and 37 °C respectively. Take the typical case of 37 °C and pH 5.5 as the example. Firstly, 100 μL of PMZS-8–Dox was taken into a cuvette and 1.9 mL MES–Tris buffer (pH 5.5, 10 mM) preheated at 37 °C was immediately added and mixed for PL measurement. The rest 2.9 mL of PMZS-8–Dox dispersion was centrifuged at 0.5 h of release, the supernatant was collected to determine the amount of released Dox by UV-vis. Then 2.9 mL of MES–Tris buffer (pH 5.5, 10 mM) preheated at 37 °C was added back to the centrifuge tube, and 100 μL of the redispersed PMZS-8–Dox was taken out again. This procedure was repeated at the time intervals of 1, 1.5, 2, 3, 4, 5, 6, 7, 10, and 24 h, and the buffer volume was gradually reduced (100 μL each repeat) from 3.0 mL to 1.8 mL after 24 h release.

Cell viability assay

Typically the murine breast carcinoma 4T1 cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich, China) at 37 °C in a 5% CO₂ atmosphere. The 4T1 cells were plated in triplicate at a density of 2000 cells per well in 96-well microplates (Costar, U.S.A.). After culture for 12 h, the cells were exposed to 0.05, 0.1, 0.5, 1, 5, 10, 20 μM of free Dox, or equivalent Dox dose of PMZS-8–Dox, or PMZS-8 nanocomposite respectively. Cytotoxicity was determined at exposure of 6, 12, 18, 24 h with a standard MTT method.

In vitro PL imaging of Dox releasing process

Briefly, 4T1 cells was seeded in a 24-well plate (Costar, U.S.A.) containing coverslips (14 mm in diameter) at a density of 50 000 cells per well, and cultured in complete medium for 24 h, and then exposed to PMZS-8–Dox (with equivalent dose of 5 μM Dox and 23 mg L⁻¹ PMZS-8), free Dox (5 μM) or PMZS-8 (23 mg L⁻¹) for 0.5, 2, 4 and 8 h respectively. 300 μL of fixing solution (4% paraformaldehyde) and 300 μL of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were added successively to each well incubating for 15 min and 3 min, and washed with PBS respectively. The coverslips were then mounted onto microscope slides and the imaging photographs were taken by reflected fluorescence microscope in three channels (bright field, excitation of band 340–390 nm and 460–495 nm) respectively. The control trial of 4T1 cells was done in parallel just without any incubation.

Results and discussion

Synthesis of PMZS nanocomposite

For getting the PMZS composite with the most thermo-sensitive PL, the reaction conditions, including polymerization temperature, amount of Mn–ZnS and Bis (NIPAm was fixed at 1.77 mmol, Table 1), were optimized. Fig. 1 displayed the temperature-responsive PL of PMZS nanocomposites and raw Mn–ZnS, and Fig. S2† showed the corresponding PL spectra. For most of PMZS composites (except PMZS-1), the PL intensity was gradually enhanced with the gradual increase of temperature from 22 to 42 °C (Fig. 1), however, the PL of raw Mn–ZnS was gradually decreased upon the increase of temperature. This comparison demonstrated that PNIPAm in PMZS composites was responsible for the enhanced PL intensity upon the increase of temperature. It is well known that PNIPAm swells at low temperature, but shrinks at high temperature. In PMZS composites, the Mn–ZnS was attached in PNIPAm, thus the gradual shrinkage of PNIPAm at the gradually increased temperature would definitely cause the gradual aggregation of Mn–ZnS and hence the enhanced PL signals of the PMZS composites.^43,44 It was worth noting that the PL of most QDs or dyes was quenched upon aggregation, but Mn–ZnS QDs had the enhanced PL upon aggregation, which made the combination of Mn–ZnS and PNIPAm the outstanding thermo-sensitive PL probes for application at body temperature.

Fig. 1 Temperature-dependent PL intensity at 600 nm of raw Mn–ZnS QDs and PMZS composites synthesized at different (a) polymerization temperatures; (b) feed amount of Mn–ZnS; and (c) feed amount of Bis. The solutions with equivalent of Mn–ZnS of 0.015 g L⁻¹ in MES–Tris buffer (pH 6.5, 10 mM) were used for PL measurement at 22–42 °C.

The polymerization temperature had great effect on the thermo-sensitive PL of PMZS (Fig. 1a). The PMZS synthesized at 37, 50 and 70 °C exhibited obvious enhanced PL response to the increase of temperature, whereas the PMZS-1 synthesized at 25 °C displayed the decreased PL with the increase of temperature (the same tendency as the raw Mn–ZnS in Fig. 1b). That was because PNIPAm formed at 25 °C had bulky and nonporous structure, which was unable to expel the water within and thus could not shrink even in the case of increased temperature. As polymerization temperature rose to 37 °C or higher, the PNIPAm had the structure of abundant micro- and nano-pores,⁴⁵ which enabled PNIPAm the great shrinkage at the increased temperature, and thus the enhanced PL of PMZS. The less thermo-sensitive PL of PMZS synthesized at polymerization temperature of 50 °C and 70 °C was ascribed to the increased tightness and inflexibility of PNIPAm at the corresponding high temperature.

The feed amount of Mn–ZnS and Bis also influenced the PL thermo-sensitivity of PMZS (Fig. 1b and c). The best thermo-sensitivity was observed at 0.65 mmol of Zn and 1.77 mmol of NIPAm (PMZS-6). The increment of Bis content led to the decreased flexibility and shrinkage of PNIPAm, and thus the reduced thermo-sensitivity of the nanocomposite (Fig. 1c). Although the thermo-sensitivity of PMZS-6 was the best, the Dox loading content and encapsulation efficiency of PMZS-6 was slightly lower than those of PMZS-8 (data were shown in the subsequent text), therefore PMZS-8 was chosen for the subsequent Dox loading and release.

Characterization of PMZS-8 nanocomposite

The characteristic bands of methylene (stretching vibration at 2922 and 2852 cm⁻¹, bending band at 1457 cm⁻¹), methyl (asymmetric stretching vibration at 2954 cm⁻¹ and bending band at 1376 cm⁻¹), amide group (1690 and 1288 cm⁻¹) and Zn–S bond (617 cm⁻¹) are observed in the FT-IR spectrum (Fig. S3†), indicating the successful combination of PNIPAm with ZnS. The HRTEM images (Fig. S4†) revealed many Mn–ZnS QDs with diameter around 5 nm were enwrapped in PNIPAm polymer. The PMZS-8 had long lifetime around 1 ms (Table S1†) and absolute quantum yield of 18.97%.

The PL of PMZS-8 was not only thermo-responsive, but also pH-responsive. As shown in Fig. 2a, the PL intensity at 600 nm of PMZS-8 in weak acidic pH (5.0–6.5) was much higher than that in neutral pH (7.0 and 7.4) at 37 °C, and the same tendency but less sensitive PL change was observed at 25 °C. This pH-responsive PL was resulted from the nature of Mn–ZnS QDs (Fig. 2b). However, the raw Mn–ZnS displayed lower PL intensities at 37 °C than at 25 °C, whereas PMZS-8 had much higher PL intensities at 37 °C than at 25 °C. This opposite tendency was resulted from the effective combination of PNIPAm and Mn–ZnS QDs, that was, Mn–ZnS QDs had the property of enhanced PL upon aggregation, while PNIPAm shrinkage at high temperature such as 37 °C (ref. 46) exactly caused the aggregation of Mn–ZnS QDs. Besides, the PMZS-8 exhibited very stable PL intensities at pH 5.5, 6.5, and 7.4 under both 25 and 37 °C (Fig. S5†), indicating the feasibility of PMZS-8 as the PL imaging probe. It was worth noting that the PL pH-responsive range of PMZS-8 happened to meet the pH difference of normal and cancer tissues, consequently the pH- and thermo-responsive PMZS-8 composite was promising for PL imaging in tumor environment owing to the much higher PL intensity at 37 °C and the weak acidic pH.

Fig. 2 PL emission at 600 nm of (a) PMZS-8 (0.06 g L⁻¹, with equivalent concentration of 0.015 g L⁻¹ Mn–ZnS) and (b) Mn–ZnS (0.015 g L⁻¹) at 25 and 37 °C in MES–Tris buffer (10 mM, pH 5.0–7.4).

PL tracking of the loaded and controllably released Dox

Owing to the swell to shrink switch of PNIPAm at low to high temperature, PNIPAm was always used for loading drugs at low temperature and releasing drugs at high temperature. Considering the drug model Dox had lower water solubility and higher hydrophobicity in neutral pH, we chose to load Dox into PMZS-8 at the low temperature (<20 °C) and in pH 7.4 MES–Tris buffer (10 mM). Under that condition, the loading content and encapsulation efficiency of PMZS-8 were 15.2% and 84.1% respectively, slightly higher than those of PMZS-6 (12.4% and 82.5%, respectively). This loading content and encapsulation efficiency values were comparable with those reported PNIPAm carriers of Dox (Table S2†),^47–51 however, the distinguishing feature of PMZS-8 over other PNIPAm carriers was the real-time PL tracking of the loaded Dox (Fig. 3) and controllably released Dox (Fig. 4).

Fig. 3 PL tracking of the loaded Dox: (a) emission of PMZS-8 (0.06 g L⁻¹) in MES–Tris buffer (pH 7.4, 10 mM) and corresponding photos under (b) sunlight and (c) UV radiation before (I) and after (II) loading of Dox.

Fig. 4 Dox release profiles in different pH (5.5, 6.5 and 7.4, MES–Tris buffer, 10 mM) at (a–c) 37 °C and (d–f) 25 °C measured by (a and d) intrinsic PL of PMZS-8; (b and e) UV-vis absorbance of Dox; and (c and f) PL-enhanced tracking of the controllably released Dox; and the photos of PMZS–Dox for the release of Dox at 37 °C in pH 5.5 MES–Tris buffer (10 mM) at time intervals of 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 10 h under (g) sunlight and (h) UV radiation.

As shown in Fig. 3, the pure PMZS-8 had high PL emission, but its PL emission was greatly quenched after loading of Dox. This quenched PL background was beneficial for the intrinsic PL tracking of the released Dox, as the subsequent enhanced PL signals in the very low background would be more sensitive to be observed. Fig. 4 showed the Dox release profiles at 25 and 37 °C and in different pHs (5.5, 6.5 and 7.4). The release profiles acquired by measuring the intrinsic PL of PMZS-8 (Fig. 4a and d) were in according with those acquired by measuring the UV-vis of the released Dox (Fig. 4b and e) respectively, and the intrinsic PL intensity of PMZS-8 was almost linearly correlative to the cumulative release amount of Dox (Fig. 4c and f). The much faster releasing and the much more cumulative release amount at 37 °C in pH 5.5 buffer were also reflected by the much faster increasing and much more greatly enhanced PL intensity of PMZS-8 (Fig. 4c vs. f). Besides, the cumulative release amount of Dox could also be directly visualized by the intrinsic PL of PMZS (Fig. 4g and h), which was more important for real-time tracking of where, when and how the Dox was released in in vitro or in vivo imagings. All these data strongly proved that the PMZS-8 could not only release the loaded Dox in the pH- and thermo-controllable manner, but also track the controllably released Dox in PL-enhanced mode.

Cell viability assay

The cytotoxicity of PMZS-8–Dox was evaluated by a standard MTT protocol using murine breast carcinoma 4T1 cell line as model, and the control groups consisted of equivalent dose of free Dox and PMZS-8 were compared (Fig. 5). PMZS-8–Dox displayed slightly lower cytotoxicity in time- and dose-dependent manner than free Dox within 24 h, and the PMZS-8 showed negligible cytotoxicity in a wide concentration range of 0.23–94 mg L⁻¹. These results indicated the biocompatible PMZS-8 was a good carrier of Dox for delayed releasing of Dox and thus inhibiting the Dox's toxicity to a certain extent.

Fig. 5 Time- and dose-dependent cytotoxicity of (a) PMZS-8–Dox; (b) free Dox, and (c) pure PMZS-8 toward murine breast cancer 4T1 cells.

In vitro PL imaging of Dox releasing process

To further prove the capability of PMZS-8 for PL-enhanced tracking of the controllably released Dox, we carried out the in vitro cell imaging experiments using the murine breast cancer 4T1 cells as model. The pictures were taken when the adherent 4T1 cells were exposed to PMZS-8–Dox (with equivalent dose of 5 μM Dox and 23 mg L⁻¹ PMZS-8, Fig. 6), PMZS-8 (23 mg L⁻¹, Fig. 7), or free Dox (5 μM, Fig. S6†) for 0.5, 2, 4 and 8 h respectively. The control experiment without any exposure was done in parallel for excluding the false signals not from the PMZS-8 and/or Dox (Fig. 6).

Fig. 6 PL imaging of Dox releasing in 4T1 cells in the absence (control) or presence of PMZS-8–Dox (equivalent dose of 5 μM Dox and 23 mg L⁻¹ of PMZS-8) for 0.5, 2, 4 and 8 h. The pictures were taken by a fluorescence microscope in bright field, under excitation of 340–390 nm (for DAPI and PMZS-8) and 460–495 nm (for Dox).

Fig. 7 PL imaging of 4T1 cells exposed to PMZS-8 (23 mg L⁻¹) for 0.5, 2, 4 and 8 h.

For the cells exposed to PMZS-8–Dox (Fig. 6), the PL signal of PMZS-8 (the orange color) around nucleus (blue color of the stained DAPI) was gradually brighten as time went on, and so was the red fluorescence of Dox. This consistence further proved the reliability of the intrinsic PL reading-out of the released Dox. Besides, the intrinsic PL of PMZS-8 could display where the drug carrier was, and when and how the Dox was released. At the exposure of 0.5 h, no orange color around nucleus was observed in either Fig. 6 or 7, suggesting that the PMZS-8–Dox or PMZS-8 had not entered the cells yet; and the faint red fluorescence of Dox in Fig. 6 (0.5 h) might arise from the tiny release of Dox in the culture medium. At time of 2 h exposure, the relatively weak orange color around nucleus in Fig. 6 and 7 revealed the existence of PMZS-8 in the cytoplasm, while the weaker orange PL in Fig. 6 than that in Fig. 7 suggested the coexistence of Dox with PMZS-8. Further prolonging the exposure times of PMZS-8–Dox resulted in the brighter and brighter orange color around nucleus (4 and 8 h, Fig. 6), meanwhile, the brighter and brighter red fluorescence was gradually shifted into the nucleus. In contrast, the cells exposed to PMZS-8 displayed nearly constant bright orange PL for 4 and 8 h (Fig. 7). This comparison demonstrated that the intrinsic PL of PMZS-8 reached saturation for 4 h, and the further PL-enhancement for 8 h exposure of PMZS-8–Dox in Fig. 6 was owing to the continuous release of Dox. For 8 h exposure, the orange PL in Fig. 6 was relatively weaker than that in Fig. 7 was ascribed to the incomplete release of Dox (Fig. 4). The slow-release of Dox by PMZS-8–Dox was proved by the weaker red fluorescence at exposure of 0.5 h in Fig. 6 than in Fig. S6.† All these results convincingly proved that the PMZS-8 could carry Dox into cytoplasm, and the intrinsic PL of PMZS-8 could self-track the controllably released Dox in the PL-enhanced mode.

Conclusions

In summary, we presented a novel multifunctional poly(N-isopropylacrylamide)–Mn–ZnS nanocomposite for the controlled release of Dox and real-time PL tracking of the released Dox in the PL-enhanced manner. Besides the thermo- and pH-responsive PL, the coupling of Mn–ZnS QDs and PNIPAm endowed the composite the much higher PL intensity at 37 °C than 25 °C and the much higher PL intensity in weak acidic pH than in neutral pH, which was promising for PL imaging in tumor microenvironment. The easily-synthesized PMZS nanocomposite was the biocompatible and smart drug carrier with the capability of self-PL tracking where, when and how the Dox was released.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21575070, 21435001, 21175073) and the Tianjin Natural Science Foundation (No. 13JCYBJC17000).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10395b

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