Smart Porous Silicon Nanoparticles with Polymeric Coatings for

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Smart porous silicon nanoparticles with polymeric coatings for sequential combination therapy Wujun Xu, Rinez Thapa, Dongfei Liu, Tuomo Nissinen, Sari Granroth, Ale Närvänen, Mika Suvanto, Hélder A. Santos, and Vesa-Pekka Lehto Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00473 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 26, 2015

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Molecular Pharmaceutics

Smart porous silicon nanoparticles with polymeric coatings for sequential combination therapy Wujun Xu,1 Rinez Thapa,2,† Dongfei Liu,3, † Tuomo Nissinen,1 Sari Granroth,4 Ale Närvänen,2 Mika Suvanto,5 Hélder A. Santos3 and Vesa-Pekka Lehto*,1 1

Department of Applied Physics, University of Eastern Finland, POB 1627, 70211 Kuopio, Finland

2

School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland

3

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki,

00014 Helsinki, Finland 4

Department of Physics and Astronomy, University of Turku, 20014 Turku, Finland

5

Department of Chemistry, University of Eastern Finland, 80101 Joensuu, Finland

†

R. Thapa and D. Liu contributed equally to this work.

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Abstract In spite of the advances in drug delivery, the preparation of smart nanocomposites capable of precisely controlled release of multiple drugs for sequential combination therapy is still challenging. Here, a novel drug delivery nanocomposite was prepared by coating porous silicon (PSi) nanoparticles with poly (beta-amino ester) (PAE) and Pluronic F-127, respectively. Two anticancer drugs, doxorubicin (DOX) and paclitaxel (PTX), were separately loaded into the core of PSi and the shell of F127. The nanocomposite displayed enhanced colloidal stability and good cytocompatibility. Moreover, a spatiotemporal drug release was achieved for sequential combination therapy by precisely controlling the release kinetics of the two tested drugs. The release of PTX and DOX occurred in a time-staggered manner; PTX was released much faster and earlier than DOX at pH 7.0. The grafted PAE on the external surface of PSi acted as a pHresponsive nanovalve for the site-specific release of DOX. In vitro cytotoxicity tests demonstrated that the DOX and PTX co-loaded nanoparticles exhibited a better synergistic effect than the free drugs in inducing cellular apoptosis. Therefore, the present study demonstrates a promising strategy to enhance the efficiency of combination cancer therapies by precisely controlling the release kinetics of different drugs.

Keywords: Porous silicon; pH-Responsive materials; Tumor microenvironment; Surface modification; Controlled drug release; Combination therapy


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Table of Contents (TOC) Graphic



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1. Introduction The utilization of nanotechnology in drug delivery has opened a new era for the development of effective carrier systems for cancer therapy. Up to now, many types of nanoparticles like polymer micelles have been studied for combination therapies.1,

2

Beyond the conventional delivery of

single drug, combination therapy, which refers to the co-administration of multiple drugs, is an increasingly important strategy to overcome undesirable side effects and to improve the survival rates in tumor therapy.1, 3-6 Generally, the combination therapy can modulate different signaling pathways in cancer cells7,

8

or improve the pharmacokinetics profile5 of anticancer drugs to

maximize the therapeutic effect and is often superior to single-drug therapy. Thus, combination therapy is now widely used as a first-line treatment in cancer patients. Compared with the healthy tissues, the abnormal metabolic mechanism of tumors produces a special surrounding microenvironment. For example, the heterogeneous and poor blood supply as well as interstitial fluid pressure (IFP) in tumors often affects the success of the cancer therapy. To overcome delivery barriers at tumors, sequential combination therapy by controlling the order of the administration drugs are widely studied. The pre-treatment with one drug sensitizes the tumor environment to the second chemotherapeutic agent, enhancing their delivery efficiency into tumor and possibly modulating different apoptotic pathways.9-12 For example, early administration of PTX is able to result in tumor cell apoptosis, expansion of interstitial space, decreasing the IFP and improving the tumor oxygenation.11 Therefore, sequential administration of PTX with other anticancer drugs has been widely used in traditional clinic neoadjuvant combination therapy.13-15 In the recent studies of nanomedicine, a new strategy of ‘tumor priming’ was developed based on sequential administration of PTX in combination therapy.11, 16, 17 PTX and a carbocyanine dye were separately loaded in two different types of polymer micelles to achieve an enhanced optical imaging of a tumor. The pre-treatment of PTX loaded polymer micelles reduced the tumor volume by 1.6fold and enhanced the near-infrared (NIR) optical signal by 2.1 fold from excised solid tumors.11


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However, even though the progress in previous studies, multiple injections and the use of multiple carriers were necessary for each drug in the sequential combination therapy, which obviously decrease patient compliance and complicate the whole therapy process. Moreover, an uncontrolled drug delivery compromised the efficiency of cancer therapy. Up to now, none of studies has described the attempts to precisely control the release of different drugs from one type of nanoparticles for sequential combination therapy.

Scheme 1. Illustration of the process to prepare the TOPSi-PAE-F127DP nanocomposite. The polymer of PAE was grafted on the surface through a Michael-type addition reaction with surface amine groups. DOX and PTX were separately loaded into the core of TOPSi nanoparticles and the external layer of F127.

Porous silicon (PSi) has attracted considerable attention due to its potential applications in drug delivery due to its high surface area, tunable surface chemistry and good biocompatibility.18-22 The goal of the present study was to design a smart drug delivery platform capable of spatiotemporal drug release, in which the release of drugs was precisely controlled to address the tumor microenvironment and enhance the efficiency of cancer therapy. PSi nanoparticles acted as the nanocarriers and were loaded with the drug DOX. The pore openings of the PSi nanoparticles were grafted with a pH-responsive nanovalve of poly(beta amino ester) (PAE). The drug PTX was encapsulated into the external layer of Pluronic F-127 ((EO)98(PO)67(EO)98) on the surface of the PSi nanoparticles (Scheme 1). These novel nanoparticles are capable of achieving the 5 ACS Paragon Plus Environment


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spatiotemporal drug release of two drugs: the encapsulated PTX is released faster and earlier under physiological conditions (pH 7.0) while the loaded DOX is confined within the pores of the PSi nanoparticles because of the PAE nanovalves. Only later, when the nanoparticles become internalized by the cancer cells, does the intracellular acidic microenvironment act as a stimulus for the nanovalve of PAE to trigger the rapid release of DOX. Based on the previous studies16, 23, it was hypothesized that the earlier released PTX would sensitize the tumor microevironment like decreasing the IFP,11 which would be beneficial to the delivery and diffusion of DOX deep into the tumor. The increase in the pO2 would also improve the cytotoxicity of an oxygen-dependent drug like DOX.23

2. Experimental Section 2.1. Preparation of the PSi Nanoparticles PSi films were produced by pulsed electrochemical etching of p+-type silicon wafers (100) with resistivity values of 0.01-0.02 Ωcm in an HF (38%)-ethanol mixture. PSi nanoparticles were prepared by ball-milling of the films in ethanol at the speed of 800 rpm for 25 min. The nanoparticles were separated by centrifuge with speed of 1500 RCF for 15 min. PSi nanoparticles in supernatant were recovered, dried at 65 °C, and further stabilized with thermal oxidation at 300 °C for 2 h. Subsequently, the nanoparticles were reacted with a solution of NH3·H2O/H2O2/H2O (volume ratio: 1/1/5) and HCl/H2O2/H2O (volume ratio: 1/1/6) at 85 °C for 15 min, respectively.24 After the reaction, the nanoparticles were rinsed three times with ethanol. The obtained sample was designated as TOPSi-OH. 2.2. Selective Surface Modification of the PSi Nanoparticles Surface amine groups are utilized to graft PAE as the nanovalve to control the release of DOX and thus the amine groups were selectively modified on the external surface leaving hydroxyl groups on the pore walls.25 The incipient-wetness impregnation method was utilized to construct the nano-


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stopper of n-hexane in the TOPSi-OH nanoparticles. After filling the mesopores with n-hexane, the nanoparticles

were

mixed

with

4.0

wt%

silane

of

3-(2-aminoethylamino)

propylmethyldimethoxysilane in dimethyl sulfoxide (DMSO) at ambient temperature. Because the pores were blocked by the nano-stoppers of n-hexane, the silane selectively reacted with the −OH groups on the external surface. The nanoparticles were rinsed with ethanol after 30 min and treated in an oven at 65 ºC for 2 h. The obtained amine-functionalized sample was designated as TOPSiNH2. 2.3. Preparation of the pH-Responsive Nanoparticles The pH-responsive nanovalve of PAE was grafted onto the external surface of the nanoparticles by using a Michael-type addition of amine groups to the diacrylates.26 Briefly, 30 mg TOPSi-NH2 nanoparticles were dispersed in 3.0 mL chloroform solution with hexamethylene diacrylate (HDA, 50 mg). The mixture was reacted at 60 °C for 1 hour in an atmosphere of N2. Subsequently, 47 mg of 4,4′-trimethylenedipiperidine (TDD) was added and the mixture was refluxed for 48 h at the same temperature. The PAE grafted TOPSi nanoparticles were recovered after centrifugation, washed with ethanol and the material was designated as TOPSi-PAE. 2.4. Preparation of DOX&PTX co-loaded nanoparticles 1.0 g of Pluronic F127 was dissolved in 4.0 mL acetonitrile containing 4 mg PTX. After evaporation of the solvent completely at 70 °C, the resulting film was hydrated with 25 ml deionized water at the same temperature to yield PTX-loaded F127 micelles.27 Meanwhile, 1 mg of DOX was dissolved in 1.0 ml ethanol with the assistance of triethylamine. The TOPSi-PAE nanoparticles were added into the DOX solution and sonicated for 30 min. The mixture was stirred at ambient temperature for 2 h. Subsequently, the DOX loaded nanoparticles were added into the F127 solution drop by drop with vigorous stirring. The resulting suspension was stirred for 4 h at 75 °C to evaporate the organic solvent and then cooled to ambient temperature. The drug-loaded nanoparticles were recovered by centrifugation, washed with H2O, and designated as TOPSi-PAE-



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F127DP. Herein the letters of ‘D’ and ‘P’ refer to the drugs of DOX and PTX, respectively. An identical protocol was used for the preparation of the control TOPSi-PAE-F127 nanoparticles except that no drugs were added during their preparation. 2.5. Preparation of Fluorescein Isothiocyanate (FITC) & DOX Co-loaded Microparticles and Nanoparticles The PSi microparticles were prepared by ball milling of PSi films at the speed of 200 rpm for 5 min. The milled particles were sieved to obtain the PSi microparticles with the diameter below 25 µm. PSi nanoparticles were prepared with the same method, as described in Section 2.1. The remaining steps for the preparation of FITC&DOX co-loaded microparticles/nanoparticles were the same as those for the preparation of DOX&PTX co-loaded TOPSi-PAE-F127 nanoparticles except that PTX was replaced with FITC. 2.6. Preparation of FITC Labelled TOPSi-NH2 Nanoparticles 20 mg of TOPSi-NH2 nanoparticles were dispersed in 5.0 mL ethanol with 1.0 mg of FITC.28 The mixture was reacted under dark for 24 h with stirring. The unreacted FITC was removed by rinsing the sample with ethanol three times. 2.7. Material Characterization Adsorption/desorption measurements were conducted using Micromeritics TriStar 3000 by N2 physisorption at 77 K. The specific surface areas of the samples were calculated using the multiplepoint Brunauer−Emmett−Teller (BET) method. The pore size distribution was calculated from the desorption branch using the Barrett−Joyner−Halenda (BJH) theory. The surface charge and hydrodynamic diameter of the nanoparticles were analyzed with a Malvern Zetasizer Nano (ZS). The surface chemistry of the samples was characterized with Fourier-transform infrared spectroscope (FT-IR, Thermo Scientific Nicolet 8700). The surface modifications were quantified with TGA (TA instruments Q50 TGA) under a N2 gas purge and CHN elementary analysis (CHNS analyzer, Vario 219 MICRO cube). The morphology of the nanoparticles was characterized with


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High-resolution TEM (HRTEM) images (JEOL JEM2100F). The FITC and DOX co-loaded microparticles were assessed using Z-stack confocal imaging in a Zeiss LSM 700 confocal laser scanning microscope (Zeiss, Germany). Fluorescent microscopy images were acquired with a 40× oil-immersion objective. Perkin-Elmer PHI 5400 was used for X-ray photoelectron spectroscopy (XPS) measurements. The photoemission spectra of C 1s, O 1s, N 1s and Si 2p together with long energy range survey spectra were collected using XPS with Mg Kα radiation (1253,6 eV). The binding energy scale was calibrated by setting the C-C/C-H component of the fitted C 1s spectra at 285 eV. The fitting procedure was done using Spectral Analysis by Curve Fitting Macro Package for Igor Pro. 2.8. In Vitro Drug Release 6.0 mg of drug loaded nanoparticles were dispersed and incubated in 1.0 mL phosphonate buffer solution (PBS) containing 0.1% Tween 80 with a pH value of 7.0 at 37 °C. At pre-determined time points, the buffer solutions suspended with nanoparticles were centrifuged. The supernatant was collected and analyzed by high-performance liquid chromatography (HPLC), as described below, in order to evaluate the released drug concentration. The obtained nanoparticles were re-dispersed in an equal volume of fresh release medium to continue the drug release tests. The release medium was changed to pH 5.0 acetic buffer solution with 0.1% Tween 80 after 4.0 hours. The same release procedure as that conducted with PBS was applied to collect samples for drug concentration analyses. The loading degree of drugs was calculated based on a similar release procedure except that only pH 5.0 buffer solution was used for prolonged time. The in vitro drug release kinetic was analyzed according to two basic empirical equations.29 In the Korsmeyer–Peppas model,

Mt/M∞=k·tn

(1)

where Mt/M∞ is the fraction of drug release, k is a kinetic constant, t is the drug release time, and n is the diffusional exponent. The drug release mechanism is based on Fickian diffusion when n ≤0.5, 9 ACS Paragon Plus Environment


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where the solvent penetration is the rate limiting step (Case I). A zero-order kinetics occurs when n=1 (Case II), which is associated with the relaxational release of loaded drug due to stresses and state-transition in hydrophilic polymers that swell in water or biological fluids. When the value of n is between 0.5 and 1, it is related to an anomalous transport (case III) where both Fickian and relaxational phenomena contribute to the drug release. Peppas and Sahlin evaluated the contributions provided by Fickian diffusion and matrix relaxation/dissolution, and ended up in the following equation:29

Mt/M∞=k1·tm+

k2·t2m

(2) where k1 is the Fickian kinetic constant and k2 is the relaxational/dissolution rate constant (i.e., anomalous transport). The coefficient m is the Fickian diffusional exponent that depends on the geometry of the particles. 2.9. HPLC Analysis of DOX and PTX The HPLC system (Shimadzu, Japan) consisted of a LC-10 AT VP pump, SIL-10 AD VP sample injector and FCV-10 AL UV/Vis detector. Chromatographic separations and subsequent quantifications of DOX and PTX were carried out at room temperature using an analytical column Inertsil ODS-3 C18, 4×150 mm (GL Sciences Inc.). The buffer A (5% ACN, 0.1% TFA) and buffer B (95% ACN, 0.1% TFA) were pumped at a flow rate of 1mL/min. The buffers were prepared freshly and degassed by sonication for 5 minutes before use. The injection volume was 100 µL and the detection wavelength was 227 nm. 2.10. Stability of Nanoparticles in PBS and Human Plasma TOPSi-NH2 and TOPSi-PAE-F127 nanoparticles were dispersed in PBS or plasma and incubated at 37 oC. The size of the nanoparticles in PBS was monitored directly at the given time points with dynamic light scattering (DLS) measurements using Zetasizer Nano (ZS). In the stability test of the nanoparticles in human plasma, the samples were diluted with H2O before analysis.30


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2.11. Fluorescence Confocal Microscope studies Suspensions of HeLA cells (400 µL, 1×105 cells/mL) were seeded in Lab-Tek Chamber Slides (Thermo Fisher Scientific, USA). After 24 h of cell attachment to the wells, the cells were rinsed three times with 1× Hank's balanced salt solution (HBSS; pH 7.4), and then the free DOX and FITC, and FITC and DOX co-loaded TOPSi-PAE-F127 (400 µL; 10 µg/mL DOX equiv.) were added into the wells. Following by 1 and 4 h incubation, the samples were removed and the wells were washed three times with 1× HBSS (pH 7.4). DAPI with a concentration of 2.8 µM was added for cell staining, as described elsewhere.31 Finally, the cells were fixed with paraformaldehyde (4.0%, w/v) in 1× PBS for 30 min at room temperature. The confocal images were taken with a Leica TCS SP5II (Leica Microsystems, Germany) inverted confocal microscope, equipped with argon (488 nm) and UV (diode 405 nm) lasers, and using a HCX PL APO 63×/1,20 W CORR/0.17 CS (water) with a water immersion micro dispenser. 2.12. Flow Cytometry Studies Suspensions of RAW 264.7 macrophages and HeLa cervical cancer cells (1.0 mL/well) were separately seeded in 12-well plates at the concentration of 1.0×106 cells/mL. Following by 24 h of cell attachment to the wells, the cells were firstly rinsed three times with 1× HBSS (pH 7.4) and then the FITC loaded TOPSi-NH2 and TOPSi-PAE-F127 nanoparticles (400 µL, 100 µg/mL) were incubated with the cells for 1 and 4 h. After washing with 1× HBSS (pH 7.4), the cells were harvested, and then fixed with paraformaldehyde (4.0%, w/v) in 1× PBS for 30 min at room temperature. Exactly 10 000 events were collected on a LSR II flow cytometer (BD Biosciences, USA) with a laser excitation wavelength of 488 nm using FACSDiva software. Untreated cells were employed as controls. 2.13. In Vitro Cytotoxicity Studies The in vitro cytotoxicities of the control TOPSi-PAE-F127 nanoparticles, the free drugs, and the drug loaded TOPSi-PAE-F127DP nanoparticles were evaluated in HeLa cell lines with the



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CellTiter-Glo assay (Promega Corporation). The free drugs or the nanoparticles were added to each well of the cell plate to achieve the predetermined concentrations and then incubated for 4 hours. After rinsing twice with 100 µL of the growth medium, the cells were further incubated at 37 °C in growth medium for 48 h. Toxicity was evaluated using CellTiter-Glo assay according to the manufacturer’s instructions. Luminescence was determined with Fluoroskan AscentFL (Thermo Labsystems). More details of the procedure can be found elsewhere.25 2.14. Statistical Analysis Results from all the tests are expressed as the mean ± s.d. for at least three independent experiments. A one–way analysis of variance (ANOVA), followed by a Student t–test was employed to analyze the data. The analysis was carried out using Origin 8.6 (OriginLab Corp., USA) and the levels of significance were set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001.

3. Results and Discussion 3.1. Preparation and characterization of the nanocomposite PAE is a type of pH-responsive cationic polymer which possesses good biocompatibility and potential applications in cancer therapy.26, 32 In the present study, PAE was polymerized onto the surface of PSi nanoparticles as nanovalves through the Michel addition reaction. However, it is difficult to use PAE solely in a drug delivery application due to its hydrophobic property in aqueous solution at pH 7.0. Pluronic F127 is a triblock polymer containing hydrophilic poly(ethylene oxide) (PEO) segments and hydrophobic poly(propylene oxide) (PPO) segments and is designated as PEO–PPO–PEO.33 The hydrophilic PEO segments are able to stabilize the hydrophobic nanoparticles in the physical medium, similar to polyethylene glycol (PEG) because of the steric hindrance, high hydrophilicity and the neutral nature of the PEO chains.34 Based on the PPO segments, F127 can form a lipophilic carrier by the encapsulation of the drug inside the hydrophobic core. Moreover, the Pluronic F127 has the potential to overcome the multidrug


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resistance in cancer therapy.27 Therefore, F127 is an ideal polymer to assemble with hydrophobic PAE on the nanoparticle to form a multifunctional nanocomposite in the present study, which both stabilizes the whole drug delivery platform and also hosts the second drug, PTX. The prepared nanoparticles were systematically characterized with several methods to investigate their physicochemical properties. The FT-IR spectra in Figure 1a show a broad peak in the region of 3700-3300 cm-1 due to the surface Si-OH groups on the TOPSi-OH sample. An intense peak in the range 1130-1000 cm-1 was assigned to Si−O−Si stretching. The peak at 1630 cm-1 was attributable to O−H bending of H2O molecules adsorbed on the sample surface. After modification with amine groups on the external surface, a peak at 1640 cm-1 appeared because of the combination of N−H bending (1650 cm-1) and O−H bending (1630 cm-1).35 A typical peak of −C=O at 1734 cm-1 was observed in the spectrum of TOPSi-PAE which indicated that the polymer PAE had been grafted onto the surface of nanoparticles. More intensive peaks of C–H stretching at 1457 cm−1 and in the range of 2800–3000 cm−1 were also found as compared to those in the sample of TOPSi-NH2. The C−N stretching appeared as a shoulder peak at 1170 cm-1 since it overlapped with the peak of Si−O−Si stretching.35,

36

. Similarly, the typical peak of –C–O stretching from the coating of

Pluronic F127 was also identified as a small shoulder peak at 1110 cm−1 in TOPSi-PAE-F127. The samples were analyzed with XPS to further confirm the surface functionalization. Figure S1 (Supporting information) shows the survey scan of XPS spectra of the samples with different surface treatments. Within the samples of TOPSi-NH2, TOPSi-PAE and TOPSi-PAE-F127, the C content gradually increased while the Si content decreased with the process of surface functionalizations (Supporting information, Table S1). The C1s spectra of the samples has been deconvoluted into different peaks for C species analysis (Table 1 and supporting information, Figure S2).37,38 The peaks of C=O (287.4 eV) and COO-R (289.0 eV) were detected in the sample of TOPSi-PAE after PAE grafting. The increase of C-O content in the sample of TOPSi-PAE-F127 also indicated the success of the F127 coating.



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Figure 1. FT-IR spectra (a) and zeta potentials (b) of the prepared nanoparticles. Errors bars represent the mean ± s.d. (n = 3).

Table 1. C species analysis from the C1s XPS spectra Relative intensity and content of chemical states of C species C-Si

C-C, C-H

C-O, C-NH

C=O, O-C-O

COO-R

(283.9 eV)

(285,0 eV)

(286.4 eV)

(287.5 eV)

(289.0 eV)

[%]

[%]

[%]

[%]

[%]

TO-NH2

4

67

29

-

-

TOP-PAE

3

77

14

2

4

TOPSi-PAE-F127

2

59

35

1

3

The surface charge of the nanoparticles was monitored to confirm the successful surface functionalization (Figure 1 b). The zeta potential of the sample TOPSi-OH was −40 mV due to surface Si−OH groups while it was changed to +36 and +42 mV for TOPSi-NH2 and TOPSi-PAE due to amine functionalization and PAE grafting, respectively. The surface charge of the TOPSiPAE-F127 decreased to +22 mV due to the neutral F127 coating on the nanoparticles. The content of the functional polymers on the surface of the nanoparticles was quantified with TGA (Figure 2a). Around 11.2±1.8% mass loss was observed for the sample TOPSi-PAE as compared to the sample TOPSi-NH2. The coating of F127 was also corroborated by comparing the mass loss (16.7±1.5%) between the TOPSi-PAE and TOPSi-PAE-F127 nanoparticles. The polymers contents 14 ACS Paragon Plus Environment

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were also calculated from elementary analysis based on the change of N element. 14.6±2.1% of PAE and 20.1±2.9% of F127 were detected in the elementary analysis, which were a bit higher values than those obtained in TGA analysis. It is probably attributed to a little amount of residual carbides in TGA analysis. Anyway, the high content of F127 on the surface of the nanoparticles was intended to improve their stability in a biological medium, which is beneficial in this type of drug delivery application.

Figure 2. TGA curves of TOPSi-NH2 and TOPSi-PAE and TOPSi-PAE-F127 (a) and Size changes of nanoparticles before (TOPSi-NH2) and after (TOPSi-PAE-F127) polymer coating in H2O (b).

Table 2. Pore parameters and plasma stability of the PSi samples

Surface area (SBET, m2/g)

Pore diameter (nm)

Pore volume (cm3/g)

TOPSi-NH2

120.1

11.8

TOPSi-PAE-F127

53.0

12.3

Sample

Diameter of samples in human plasma (nm) t= 0 min

t= 15 min

0.46

186±10

1411±87

0.27

252±16

336±29

The size change of the nanoparticles before and after polymer coating was monitored by DLS analysis. After coating with PAE and F127, the diameter of the TOPSi-PAE-F127 nanoparticles increased from 185±10 nm to 252±16 nm (Figure 2b). The polymer coating was also observed on



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TOPSi-PAE-F127 with TEM analysis as compared with the control sample TOPSi-NH2 (Supporting information, Figure S3).

Figure 3. Size stability of nanoparticles in PBS at 37 °C. Error bars represent the mean ± s.d. (n=3)

As shown in Scheme 1, F127 possesses hydrophilic PEO segments which have the ability to inhibit aggregation, reduce protein adsorption, and prevent recognition by the reticuloendothelial system (RES).33, 39 Thus, the stability of the nanocomposite TOPSi-PAE-F127 was evaluated in PBS and in human plasma solutions. The results shown in Figure 3 indicate that the shell layer of F127 on the TOPSi-PAE-F127 enhanced its colloidal stability in the buffer solution. The size of the nanoparticles slightly increased from 252±16 to 285±24 nm in the first 15 min due to a salt effect and further increased to 391±35 nm by 90 min (Figure 3). However, the diameter of TOPSi-NH2 without F127 coating increased sharply from 185±10 to 945±25

nm within 15 min and it was

around 1.7±0.1 µm when the incubation time prolonged to 90 min. Similar results were obtained when the samples were incubated in human plasma (Table 2). The F127 shell layer successfully decreased the protein adsorption and thus the sample of TOPSi-PAE-F127 was much more stable in plasma than TOPSi-NH2. The size increase was still observed in 15 min because some of the plasma proteins were adsorbed on the positively charged surface of the TOPSi-PAE-F127 nanoparticles. 16 ACS Paragon Plus Environment

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The porous parameters of the nanocomposite before and after polymer coating were obtained from N2 ad/desorption measurements. Both of the samples presented the characteristic type-IV isotherm of mesoporous materials (Supporting information, Figure S4).40 The surface area and pore volume of TOPSi-PAE-F127 decreased while the pore diameter increased to some extent, in comparison to the corresponding values of TOPSi-NH2 (Table 2 and Supporting information, Figure S5). The coated polymers had solidified to block a part of the small pores when the samples were analyzed at 77 K. 3.2. Drug Loading and in Vitro Drug Release

Figure 4. In vitro release curves of drugs from DOX&PTX co-loaded TOPSi-PAE-F127DP nanoparticles. All release experiments were conducted at 37 ° C. Error bars represent the mean ± s.d. (n=3)

The loadings of DOX and PTX on PSi are based on the adsorption and hydrophobic interaction, respectively. The poly (beta amino ester) (PAE) chains on the PSi nanoparticles were extended or swelled in the loading solution of DOX/ethanol and thus the pores are opened for the easy access of the cargo molecules. Because PSi has highly porous structure, DOX was adsorbed into the pores of PSi, and the process was driven by the concentration gradient. PTX is a hydrophobic molecule and it has poor solubility in aqueous solution. Pluronic F127 has hydrophilic poly(ethylene oxide) (PEO) segments and hydrophobic poly(propylene oxide) (PPO) segments in its triblock structure.



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Therefore, PTX was loaded into the hydrophobic PPO core of F127 micelles due to the hydrophobic interaction after the mixture of TOPSi-PAE-F127 and PTX was hydrated. The loading degree of DOX and PTX in the nanoparticles was 1.2 and 1.8 wt%, respectively, which was calculated based on the released drug concentration. FITC&DOX co-loaded TOPSi-PAE-F127 microparticles were prepared and imaged in a fluorescent microscope to identify the location of the drugs. FITC is a type of fluorescent molecule which possesses similar physical properties as PTX. Both compounds are poorly soluble in aqueous solutions but they are freely soluble in ethanol. Therefore, it was decided to substitute the non-fluorescent PTX with FITC in experiments for imaging the location of different cargos in the sample. PSi microparticles were used due to the optical resolution limitations of the fluorescent microscope. The top view image indicates that the sample had a yellow color because of the overlap of green and red colors, corresponding to the emissions of FITC and DOX, respectively (Supporting information, Figure S6a). Along the depth increase shown in the Z-axis, a red color originating from the core of the microparticles was gradually observed (Supporting information, Figure S6b, c). In addition, the yellow color was still around the sample. These results confirmed that DOX and FITC (PTX) had been individually loaded into the core and shell of TOPSi-PAE-F127DP. The in vitro drug release from the co-loaded TOPSi-PAE-F127DP nanoparticles was evaluated in buffer solutions at different pH values. For investigating the spatiotemporal characteristics of the controlled drug release behavior, the nanoparticles were initially immersed in PBS at pH 7.0 for 4.0 h and then the release medium was changed to pH 5.0 to simulate the acidic environment inside a cancer cell. The release of PTX and DOX shown in Figure 4 occurred in time-staggered and sitespecific manners, i.e. there was spatiotemporally controlled drug release. At pH 7.0, the release of DOX was very slow while the release of PTX was significantly higher under the same conditions. Around 8 % of DOX and 56 % of PTX were released during the first 4 h at pH 7.0. Nevertheless, a burst release of DOX was observed and almost 80 % of DOX had been released in 2 h when the


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release medium was changed to pH 5.0 acetic acid buffer solution. The significantly faster release of PTX at pH 7.0 ensured that the releases of PTX and DOX occurred in a time-staggered manner. Based on the previous studies

11, 23

, the PTX released early reduces the IFP and improves the

oxygenation within the tumor, enhancing the penetration of nanoparticles and diffusion of anticancer drugs deep into the tumor. The spatiotemporally controlled drug delivery of DOX and PTX can be attributed to the different release mechanisms. The release of DOX was controlled by the pH-responsive nanovalves PAE grafted on the surface of nanoparticles. It formed a dense hydrophobic layer on the surface of the nanoparticles to block the pore openings and thus preventing the release of DOX at pH 7.0. On the other hand, the nanovalves of PAE become hydrophilic and swollen rapidly due to the ionization of the amine groups under acidic conditions (pH0.5) from the Korsmeyer–Peppas model, which indicates that both Fickian (k1) and relaxational phenomena (k2) contribute to the drug release process. Table 3. In-vitro release kinetics parameters of DOX and PTX at pH 7.0 and 5.0 pH

Drug

Korsmeyer–Peppas model Peppas-Sahlin model k n r2 k1 k2 m** r2** 7.0 DOX 0.064 0.243 0.983 0.086 -0.018* 0.39 1.0 PTX 0.305 0.452 0.971 0.271 0.003 0.39 0.990 5.0 DOX 0.569 0.210 0.994 0.786 -0.207* 0.39 0.999 PTX 0.314 0.548 0.987 0.202 0.109 0.39 0.994 *The negative values obtained for k2 are interpreted in terms of a relaxation process insignificant compared to the diffusion mechanism.41 ** m relates on the geometry of the particles. It was found that 0.39 was the optimal for the PSi nanoparticles; r2, correlation coefficient

3.3. Cell Uptake and Intracellular Distribution Flow cytometry was used to evaluate the uptake of nanoparticles into HeLa and RAW 264.7 macrophage cells. FITC labelled TOPSi-PAE-F127 and TOPSi-NH2 nanoparticles were tested in the study. The obtained histograms shown in Figure 5 indicate that the interaction between the PSi nanoparticles and the cells increased as a function of incubation time, independent on the types of PSi nanoparticles and types of tested cancer cells. Moreover, the internalization of the TOPSi-PAEF127 nanoparticles in HeLa and RAW 264.7 macrophage cells was siginificantly higher than that of the TOPSi-NH2 nanoparticles, indicating that the nanoparticles may have high efficiency in cancer therapy. The cellular uptake of nanoparticles are affected by several factors like surface functionalization and particle stability. Based on the previous reports,42,43 the presence of PAE can


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greatly promote the cellular uptake of the cargos. Moreover, the size of TOPSi-NH2 is not stable in cell medium. The TOPSi-NH2 aggregated extensively in PBS in 0.5 h, which also hindered the internalization of TOPSi-NH2. Therefore, the TOPSi-PAE-F127 nanoparticles have higher cellular uptake than TOPSi-NH2 even though the sample has the external coating of F127.

Figure 5. Flow cytometry histograms (left column) and mean fluorescence intensity (MFI, right column) of different nanoparticles incubated with HeLa (a and b) and RAW264.7 (c and d) cells for 1 h (a and c) and 4 h (b and d). Flow cytometry histograms show the fluorescent intensity of the control cells, cells incubated with FITC labelled TOPSi-NH2 (I) and FITC&DOX co-loaded TOPSi-PAE-F127 (II). For the ﬂow cytometry analysis, the MFI values of TOPSi-PAE-



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F127 samples were compared with TOPSi-NH2. Errors bars represent the mean ± s.d. (n = 3). The level of significance were set at a probability of ***p˂0.001.

The intracellular distribution behavior of the nanoparticles and their payloads were investaged with HeLa cells by confocal microscopy. To image the intracellular distribution of drugs, the FITC&DOX co-loaded TOPSi-PAE-F127 nanoparticles were analyzed and the mixture of free FITC and DOX

Figure 6. Confocal images of free DOX, FITC, and FITC&DOX co-loaded TOPSi-PAE-F127 that incubated with HeLa cells for 1 and 4 h.


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served as the control. As shown in Figure 6, free DOX and FITC are able to be internalized and accumulated inside cells. A homogenous weak green (FITC) and red (DOX) fluorescences was observed in the nucleis of cells after 1 h incubation with free FITC and DOX. The fluorescence intensity was increased with the prolongation of incubation time to 4 h. However, when the cells incubated with the co-loaded nanoparticles, the stronger green and red fluorescences were distributed widely in the cytoplasm as compared with the free DOX and FITC, suggesting that the co-loaded nanoparticles were successfully internalized by tumor cells via endocytosis. The acidic microevironments in endosome and cytosome triggered the quick release of the loaded drugs once the nanoparticles were internalized. The layer of Pluronic F127 was simutaneously released from the nanoparticles, which is able to inhibit the drug efflux transporters in cancer cells.27 Therefore, it is expected that higher concentration of drugs are delivered inside the cancer cells after the incuation with the co-loaded nanoparticles. 3.4. Cytotoxicity Studies As compared with the untreated HeLa cells, incubation with the control TOPSi-PAE-F127 nanoparticles did not show any clear evidence of cytotoxicity even when the concentration was as high as 400 µg/mL (Figure S8), demonstrating that the TOPSi-PAE-F127 nanoparticles possess good cytocompatibility. To verify the synergistic effect of the drug-loaded TOPSi-PAE-F127DP, HeLa cells were incubated with either free drugs or drug-loaded nanoparticles, and cell viability was measured with a luminescent CellTiter-Glo assay. The dose-dependent cell cytotoxicity behavior and the synergistic effect of the drug combination are shown in Figure 7. The free drugs combination (DOX+PTX) led to higher cytotoxicity than either of the free single drugs (DOX or PTX), because the combination of free drugs affected different signaling pathways within the cancer cells.31 Moreover, the nanocomposite of TOPSi-PAE-F127DP exhibited the highest activity in the inhibition of cell proliferation. However, in view of the higher toxicity of the free drugs to healthy tissues because of their non-specific distribution and uncontrolled diffusion rates, the drug-



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loaded nanoparticles would be clearly preferable for use in combination therapy. The spatiotemporal release of PTX and DOX from the TOPSi-PAE-F127DP nanoparticles not only induced a more therapeutically relevant synergistic effect in a time-staggered manner as compared to free drugs combination, but also increased the local intracellular drug concentrations because of the pH-responsive site-specific drug release.

Figure 7. In vitro cytotoxicity of the untreated cells as a negative (Neg.) control, free DOX, free PTX, free drugs of DOX+PTX, and the drugs loaded TOPSi-PAE-F127DP nanoparticles against HeLa cells (mean ± SD, n = 4). The results were normalized to untreated cells, and 0.1% Triton X-100 was used as a positive control. Toward the HeLa cell viability, the drugs loaded TOPSi-PAE-F127DP nanoparticles are compared with free drugs of DOX+PTX; the level of significance was set at a probability of * p < 0.05 and ** p < 0.01.

4. Conclusions A smart delivery platform with spatiotemporal drug release has been successfully developed for sequential combination therapy. The release characteristics of DOX and PTX were precisely 24 ACS Paragon Plus Environment

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controlled according to their different mechanisms of action. The early release of PTX is anticipated to sensitize the tumor microenvironment for overcoming the delivery barriers for DOX. A pHresponsive release of DOX was achieved which is beneficial in site-specific cancer therapy to minimize the drug side effects and thus therapeutic efficiency can be enhanced. Moreover, the layer of Pluronic F127 has the potential to overcome the drug resistance in cancer cells. Therefore, thesmart nanocomposite developed represents a novel attractive drug delivery carrier device to address the tumor microenvironment by sequential combination therapy. Future research will focus on the evaluation of the in vivo antitumor efficiency potential of these smart nanoparticles. 5. Notes The authors declare no competing financial interest. Acknowledgments The study has been funded by the strategic funding (NAMBER) and the postdoc research funding of the University of Eastern Finland. The authors thank SIB Labs, University of Eastern Finland for providing laboratory facilities. Dr. H.A. Santos acknowledges financial support from the Academy of Finland (grants no. 252215 and 281300), the University of Helsinki Research Funds, the Biocentrum Helsinki, and the European Research Council (FP/2007–2013, grant no. 310892).

Supporting Information This information is available free of charge via the Internet at http://pubs.acs.org/.

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