Raspberry-Shaped Independent Temperature and pH Dual

Subscriber access provided by UNIV LAVAL

Research Note/Communication

Raspberry-shaped independent temperature and pH dualresponsive CPMAA@CPNIPAM yolk/shell microspheres for site-specific targeted delivery of anti-cancer drugs Lei Liu, Jinshan Guo, and Peng Liu Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b05004 • Publication Date (Web): 05 Apr 2016 Downloaded from http://pubs.acs.org on April 7, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Industrial & Engineering Chemistry Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Industrial & Engineering Chemistry Research

Raspberry-shaped independent temperature and pH dual-responsive CPMAA@CPNIPAM yolk/shell microspheres for site-specific targeted delivery of anti-cancer drugs Lei Liu, Jinshan Guo, Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT:

Raspberry-shaped

CPMAA@CPNIPAM

yolk/shell

microspheres

(RS-

CPMAA@CPNIPAM), with independent pH sensitive movable crosslinked poly(methacrylic acid) (CPMAA) cores and temperature responsive crosslinked poly(N-isopropylacrylamide) (CPNIPAM) shells, have been successfully fabricated as potential drug delivery system (DDS) for the controlled release of anticancer drugs, via the one-pot two-step “self-removing” approach based on the consecutive radical seeded emulsion copolymerization. Their formation mechanism was also proposed and the hydrogen bonds between amide groups (in NIPAM and its oligomers) with carboxyl groups (in CPMAA) should be the determining factor for the different morphology. Compared with the regular CPMAA@CPNIPAM core/shell microspheres, the RSCPMAA@CPNIPAM yolk/shell microspheres possessed higher drug-loading capacity and better

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 22

controlled release performance, with Doxorubicin (DOX) as a model anti-cancer drug. Most importantly, the novel raspberry-shaped yolk/shell microspheres possessed excellent site-specific targeted release performance, with minimal drug leakage during the body circulation.

Keywords: drug delivery system; yolk/shell nanoparticles; dual-stimuli responsive; controlled release; “self-removing” approach

INTRODUCTION Stimuli-responsive core/shell polymeric microspheres have attracted more and more research interest as drug delivery system (DDS) in the last decades due to their unique structure.1 Especially for those with the multi-stimuli responsiveness, such as pH, temperature and redox, in which physiological indexes there are significant differences between the normal tissues and tumor tissues,2 enormous efforts have been dedicated to develop intelligent DDS for the ondemand site-specific delivery of anti-cancer drugs in cancer chemotherapy in the last decades. The temperature and pH dual-responsive ones might be the promising DDS for anti-cancer drugs, with the pH responsive cores for the drug-loading and pH triggered release and the temperature responsive shells as on-off gate to modulate the drug release performance.3 Most recently, it has been reported that the hollow layer between the core and shell materials in the yolk/shell microspheres would provide enough space for the volume expansion the core materials during drug-loading, thus high drug-loading capacity could be achieved.3 Also due to the hollow layer, the interference each other in the volume transition of both the core and shell materials could be efficiently avoided4, thus the yolk/shell microspheres show independent multi-responsive property.5


2

Page 3 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For the fabrication of the yolk/shell polymeric microspheres, an easily removable middle layer is needed, such as SiO2, which could be etched with hydrofluoric acid to introduce the hollow middle layer.6-8 Wu and Weda developed a facile “one-pot ‘self-removing’ process” for the temperature-responsive hollow microspheres, in which the formation of the shells and dissolution of the cores are fulfilled in the same aqueous condition, by using a thermo-sensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), as a reversible template without the need of further calcination or chemical etching.9,10 Although the multi-layered core/shell microspheres with SiO2 middle layer is universal for most of the monomers and their polymers, the “one-pot ‘self-removing’ process” possesses the unique advantages for the temperature-responsive hollow microspheres, due to its simple and convenient manipulation.

NIPAM DVB APS SDS

APS SDS

DVB, APS and SDS

CPMAA@PNIPAM growing and connecting

washing with water at r.t.

CPMAA@CPNIPAM

RS-CPMAA@CPNIPAM CPMAA@PNIPAM@CPNIPAM : CPMAA core

: NIPAM

:CPNIPAM nucleus

: linear PNIPAM : CPNIPAM nanogel

Scheme 1. Formation mechanism of the RS-CPMAA@CPNIPAM yolk/shell microspheres.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 22

In the present work, the technique was used for the first time to design raspberry-shaped CPMAA@CPNIPAM yolk/shell microspheres (RS-CPMAA@CPNIPAM) with independent temperature and pH dual-responsive property and their formation mechanism was also proposed (Scheme 1). As potential DDS for the controlled release of anticancer drugs, their drug-loading and stimuli-responsive controlled release performance was compared with the regular CPMAA@CPNIPAM core/shell microspheres.

EXPERIMENTAL SECTION Materials and reagents. Methacrylic acid (MAA, Tianjin Chemical Reagent II Co.) was purified by vacuum distillation before use. N-Isopropylacrylamide (NIPAM, Aldrich) was obtained from Aldrich and re-crystallized from n-hexane. Ethylene glycol dimethacrylate (EGDMA) was purchased from Aldrich and used without any purification. Divinyl benzene (DVB) (analytical reagents, Tianjin Chemicals Co. Ltd., China) was used as received without any further treatment. Ammonium persulfate (APS, Tianjin Chemicals Co. Ltd., China) was re-crystallized from ethanol before use. Azobisisobutyronitrile (AIBN, AR, Tianjin Chemical Co. Ltd.) were recrystallized twice from methanol before use. Doxorubicin (DOX) was obtained from Beijing Huafeng Lianbo Technology Co. Ltd., Beijing, China. Sodium dodecyl sulfate (SDS) and other regents were of analytical grade and used without any further treatment. Double distilled water was used throughout.

CPMAA nanoparticles. The CPMAA nanoparticles were prepared by the distillation copolymerization with MAA as monomer, EGDMA as cross linker, AIBN as initiator. A typical procedure was as follows: MAA (3.0 mL), EGDMA (2.0 mL) and AIBN (0.10 g) were dissolved into 200 mL neat acetonitrile in a dried 250 mL two-necked flask. The two-necked flask


4

Page 5 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


attaching with a Vigreux column, Liebig condenser and receiver was submerged in a heating mantle. The reaction mixture was heated from room temperature to boiling state in 30 min. Then the reaction was carried out with distilling the solvent out of the reaction system and ended after 40 mL acetonitrile had been distilled off the reaction mixture in 90 min. After the reaction, the resultant CPMAA nanoparticles were purified by repeating centrifugation, decantation and resuspension in acetonitrile for three times, finally dried in a vacuum oven in 50 °C until a constant weight.

Yolk/shell

RS-CPMAA@CPNIPAM

microspheres.

The

raspberry-shaped

CPMAA@CPNIPAM yolk/shell microspheres (RS-CPMAA@CPNIPAM) were synthesized by “self-removing” approach based on the consecutive radical seeded emulsion copolymerization. 0.2 g CPMAA cores were dispersed into 45 mL deionized water in 100 mL three-necked flask, 0.4 g NIPAM and 2 mg SDS were added into the flask. The mixture was heated up to 70 °C after degassing under purging nitrogen. After 0.5 h, 0.05 g APS with 5.0 mL H2O was added into initiate the polymerization. The reaction was continued for 2 h with continuous stirring. Then 0.2 g NIPAM, 0.50 mL DVB and 0.05 g APS with 5 mL H2O were added into the mixture. After reaction continuing 4 h, the mixture was cooled to room temperature. Then, the resultant microspheres were purified by repeated centrifugation, decantation and re-suspension in deionized water three times. To demonstrate the formation mechanism and avoid the dissolution of the uncrosslinked PNIPAM layers, the intermediate products, the CPMAA@PNIPAM microspheres before the fabrication of the CPNIPAM shells, and the sandwich structured CPMAA@PNIPAM@CPNIPAM microspheres after polymerization for 0.5 h and 1.0 h, were fixed in ethanol for the morphological analysis with TEM technique.


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 22

For comparison, the regular CPMAA@CPNIPAM core/shell microspheres were prepared by the radical seeded emulsion copolymerization technique, with the CPMAA nanoparticles as seeds. In a typical route, 0.2 g CPMAA cores were dispersed in 45 mL deionized water in 100 mL three-necked flask, 0.2 g NIPAM, 0.5 mL DVB and 2 mg SDS were added into the flask. The mixture was heated up to 70 °C after degassing under purging nitrogen. After 0.5 h, 0.05 g APS with 5.0 mL H2O was added into initiate the polymerization. The reaction was continued for 4 h with continuous stirring. The resultant microspheres were collected with the same procedure above.

Drug-loading and controlled release. 10.0 mg dual-stimuli responsive microspheres were ultrasonically dispersed into the 6 mL of 1.0 mg mL-1 DOX solution and then the solution was adjusted to pH 5.0 or 6.5 or 7.4. After sharking for 48 h in the dark, the DOX-loaded microspheres were separated by centrifugation. The DOX concentration in the supernatant solution was analyzed with UV−vis spectrophotometer at its maximum absorbance of 480 nm. The drug-loading capacity (DLC) was expressed as the mass ratio of the DOX loaded and the microspheres. Then the DOX-loaded microspheres were diluted to 10.0 mL pH=5.0 or 7.4 phosphatebuffered saline (PBS), and transferred into dialysis tubes with a molecular weight cutoff of 14000 and immersed into 140 mL of the PBS with the same pH value at 37°C or 25°C, respectively. 5.0 mL of the solutions were taken out at certain time intervals to measure the drug concentrations in the dialysates with UV-vis spectrometry. Then, 5.0 mL fresh solution with the same pH value was added after each sampling to keep the total volume of the solution constant. The cumulative release rate was calculated with the following equation:11


6

Page 7 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cumulative release (%) = Mt/Mtotal × 100%

(1)

where Mt is the mass of the released drug in the releasing medium at any time and Mtotal is the initial total mass of the drug loaded in the microspheres. The Korsmeyer-Peppas model, a simple semiempirical equation, was also utilized to establish a drug release mechanism of the present system, with the following equation:11,12 Mt/M∞ = k⋅tn

(2)

where Mt and M∞ are the amount of the drug released at time t and the maximum amount of the drug released, respectively; k is the characteristic constant; and the exponent n describes the type of diffusion.

Analysis and characterization. The morphology and size of the nanoparticles and microspheres were determined by transmission electron microscopy (TEM) using a JEM1200 EX/S microscope (JEOL, Tokyo, Japan). The samples were dispersed in deionized water and a drop of the dispersion was dropped onto the surface of a copper grid covered with a carbon membrane. Elemental analysis of the microspheres was performed on an Elementar vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). The hydrodynamic diameters of the nanoparticles and microspheres were analyzed via dynamic light scattering (DLS, BI-200SM) using deionized water as the solvent. Scattered light was collected at a fixed angle of 90° for duration of ~5 min. The Fourier transform infrared (FT-IR) spectra were recorded with a Bruker IFS 66 v/s infrared spectrometer in 400−4000 cm−1 with a resolution of 4 cm−1 as KBr pellets.


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 22

UV-vis absorption spectra were measured on a TU-1901 UV-Vis spectrometer with a laser source of wavelength at 233 nm as an excitation source for the determination of the concentration of DOX.

RESULTS AND DISCUSSION Preparation

of

yolk/shell

RS-CPMAA@CPNIPAM

microspheres.

The

CPMAA

nanoparticles were prepared as the pH responsive core materials via the distillation precipitation polymerization of MAA. They were spherical in shape and monodisperse in size, with an average diameter of 51.5 nm (Figure 1a). A strong absorbance peak at 1727 cm-1 corresponding to the carbonyl groups could be seen in their FT-IR spectrum (Figure 2). Due to the plentiful carboxyl acid groups in the CPMAA nanoparticles, which could deprotonate in the media with pH values higher than its pKa of 4.4,13 they showed obvious pH responsive property, the hydrodynamic diameter (Dh) increased with polydispersity of 0.163~0.305 when increasing the media pH, because of the electrostatic repulsive force between the deprotonated specie, namely carboxylate anions (Figure 3a).5


8

Page 9 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. TEM images of (a) CPMAA cores, (band d) core/shell CPMAA@CPNIPAM, (c and e) yolk/shell RS-CPMAA@CPNIPAM microspheres, and the intermediate products fixed in ethanol: CPMAA@PNIPAM microspheres (f), CPMAA@PNIPAM@CPNIPAM microspheres after polymerization for 0.5 h (g) and 1.0 h (h).

After the one-pot two-step “self-removing” seeded emulsion polymerization, the strong absorbance peaks at 1655 cm-1 (C=O stretching, amide I), 1537 cm-1 (N-H bending, amide II) and doublet peaks at 1367 and 1387 cm-1 (C-H bending, isopropyl) in the FT-IR spectra of the two microspheres revealed the successful coating of the CPNIPAM shells onto the CPMAA cores (Figure

2)14.

With

the

one-step

seeded

emulsion

copolymerization,

the

regular

CPMAA@CPNIPAM core/shell microspheres with average diameter of 85.5 nm and shell thickness of 17.0 nm were prepared (Figure 1b), along with very few nanoparticles of about 30


9


nm resulting from the nucleation which is isolated from the CPMAA seeds in the seeded emulsion polymerization.15 However, the raspberry-shaped CPMAA@CPNIPAM yolk/shell microspheres (RS-CPMAA@CPNIPAM) with average diameter of 116.3 nm were obtained via the

“self-removing”

approach

based

on

the

consecutive

radical

seeded

emulsion

copolymerization (Figure 1c), although they showed the similar FT-IR spectrum as the CPMAA@CPNIPAM core/shell microspheres. However, the inner diameters of the CPNIPAM shells were about 77 nm and 82 nm for the regular CPMAA@CPNIPAM core/shell microspheres and RS-CPMAA@CPNIPAM yolk/shell microspheres (Figure 1 d and e) respectively, much bigger than the diameter of the CPMAA cores. The phenomena demonstrated that the core/shell structure of these two microspheres must be different from that in the common yolk/shell microspheres.

90 RS-CPMAA@CPNIPAM

75 CPMAA@CPNIPAM

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

1387 1367

60 45

1655

1537

CPMAA

30 15 0 4000 3500 3000 2500 2000 1500 1000

500

-1

Wavenumber (cm )

Figure 2. FTIR spectra of the CPMAA cores, core/shell CPMAA@CPNIPAM and yolk/shell RS-CPMAA@CPNIPAM microspheres.


10

Page 11 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The strong hydrogen bonds between amide groups (in NIPAM and its oligomers) with carboxyl groups (in CPMAA) might be the main factor for the different morphologies.16 The difference in NIPAM concentration in the formation of the CPNIPAM shells might be the other reason. The N content in the RS-CPMAA@CPNIPAM yolk/shell microspheres was determined to be 4.60%, much higher than that in the CPMAA@CPNIPAM core/shell microspheres of 2.36%, revealing the composition difference between the two microspheres. For the regular CPMAA@CPNIPAM core/shell microspheres, NIPAM and its oligomers could form hydrogen bonds with the CPMAA cores, so they could be adsorbed onto the CPMAA cores, and then grew on the surface of the CPMAA cores achieve the uniform CPNIPAM shells, except for the CPNIPAM nanogels formed from the excess NIPAM.15 As for the one-pot two-step “self-removing” seeded emulsion polymerization, the PNIPAM shells were formed onto the CPMAA cores to form the CPMAA@PNIPAM core/shell microspheres in the first step of polymerization. The formation mechanism is similar as the regular CPMAA@CPNIPAM core/shell microspheres abovementioned, except the crosslinking. Just due to the uncrosslinked structure of the PNIPAM shells, there is no distinct borderline between the CPMAA cores and the PNIPAM shells, as shown in Figure 1 f. It’s worth noting that the residual NIPAM monomer would participate in the second step of polymerization. Therein, the CPMAA cores were encapsulated by the PNIPAM shells, it impeded the formation of the hydrogen bonds between amide groups (in NIPAM and its oligomers) with carboxyl groups (in CPMAA). So the primary CPNIPAM nuclei were formed in the solution with the higher NIPAM concentration, and then the CPNIPAM nanogels were adsorbed onto the CPMAA@PNIPAM core/shell microspheres, grew and linked each other sequentially to form the raspberry-shaped CPMAA@PNIPAM@CPNIPAM sandwich structured microspheres (Scheme 1), as shown in Figure 1 g and h. Please note that the particle sizes of the microspheres in Figure 1 f-h are not


11


comparable with others, because the three samples were fixed with ethanol in order to avoid the dissolution of the PNIPAM layers, while other samples were naturally dried. After cooling to the room temperature, the middle layer of the linear PNIPAM in the raspberry-shaped CPMAA@PNIPAM@CPNIPAM sandwich structured microspheres was dissolved and removed off. It must result into a hollow layer between the CPMAA cores and the CPNIPAM shells. Thus the product should be the yolk/shell structure, although the hollow layer could not be seen directly from TEM analysis due to the high contrast of the CPNIPAM shells formed by interconnecting of the DVB-crosslinked CPNIPAM nanogels with relatively high crosslinking degree.

280

400

a

b

380 360

240 340

Dh (nm)

Dh (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 22

200

160

320 300 280 260 240

120 4

6

8 pH

10

12

220 24

CPMAA@CPNIPAM RS-CPMAA@CPNIPAM

26

28

30 32 34 Temperature (C)

36

38

Figure 3. The average hydrodynamic diameters of the CPMAA cores in different pH values at 25 °C (a) and the average hydrodynamic diameters of the CPMAA@CPNIPAM microspheres in different temperatures at pH=7.0 (b).

In spite of the different morphologies, the two kinds of CPMAA@CPNIPAM microspheres showed the similar temperature responsive characteristic from the DLS analysis (Figure 3b), although the RS-CPMAA@CPNIPAM yolk/shell microspheres showed the bigger hydrodynamic diameter (Dh), as well as the bigger particle size in the TEM analysis, due to the CPNIPAM


12

Page 13 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shells were fabricated in the solution with higher NIPAM concentration for the RSCPMAA@CPNIPAM yolk/shell microspheres. The shrinking percentage at 37 °C, denoted as the volume ratio of the swollen microspheres at 37 °C and 25 °C ((Dh(nm)37°C/ Dh(nm)25°C)3), were calculated from the DLS results to be 0.37 and 0.36 for the regular CPMAA@CPNIPAM core/shell microspheres and RS-CPMAA@CPNIPAM yolk/shell microspheres respectively. Although the inner diameters of the CPNIPAM shells in the RS-CPMAA@CPNIPAM yolk/shell microspheres (Figure 1 e) was bigger than that in the regular CPMAA@CPNIPAM core/shell microspheres (Figure 1), their shrinking percentages at 37 °C were similar, might be due to the CPNIPAM shells in the RS-CPMAA@CPNIPAM yolk/shell microspheres were constructed with the crosslinked PNIPAM nanogels, as shown in Scheme 1.

Drug-loading and stimuli-responsive controlled release. As potential DDS, the drug-loading capacity (DLC, mass ratio of loaded drug and carrier) was measured by dispersing 10 mg microspheres in 6.0 mL 1.0 mg/mL Doxorubicin (DOX) solution at pH 5.0, 6.5 or 7.4 for 48 h. After the DOX-loaded microspheres were separated, the DOX concentration in supernatant liquid was detected by UV−vis spectrophotometer to calculate the DLC of the two microspheres, as summarized in Table 1. For both drug carriers, the DLC increased with the increasing of the pH value of the drug-loading media. In the three media, the carboxylic acid groups of the CPMAA cores deprotonate into the carboxylate anions, and the negative charges increase with increasing of the media pH. This is beneficial to the drug-loading via electrostatic interaction. On the other hand, as a weak base with pKa of 8.30,17 DOX protonates in the three media. Increasing the media pH leads to a decrease of the protonated DOX, going against the drug-loading. The drug-loading via electrostatic interaction is a compromise between the two opposite factors.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

There are also other factors affecting the drug-loading, besides the two ones abovementioned. The CPMAA cores swelled more obviously in higher pH media due to the deprotonation (Figure 3a), making it easier for the diffusion of drug molecules into the drug carriers. And the DOX solubility decreased with increasing the media pH, also favoring the drug-loading. It is interesting to find that the DLC of the RS-PMAA@CPNIPAM yolk/shell microspheres is higher than that of the CPMAA@PNIPAM core/shell microspheres in all three cases, especially for that at pH 7.4. Although the two microspheres were prepared in aqueous system, in which the CPMAA cores were swollen, their volume expansion behaviors during the drug-loading must be different. For the CPMAA@PNIPAM core/shell microspheres, the CPNIPAM shell would limit the volume expansion of the CPMAA cores in drug-loading.4 The middle layer of the linear PNIPAM of the raspberry-shaped CPMAA@PNIPAM@CPNIPAM sandwich structured microspheres was dissolved to leave a low-density transition layer between the CPMAA cores and the CPNIPAM shells in the RS-CPMAA@CPNIPAM yolk/shell microspheres. The lowdensity transition layer could provide space for the volume expansion of the CPMAA cores in drug-loading, although it could not be intuitively seen in the TEM image due to the irregularly shaped CPNIPAM nanogels in the CPNIPAM shells (Figure 1c). Besides, DOX could also be loaded onto the CPNIPAM shell via hydrogen bonds.18

Table 1. DLC of the two drug carriers at different pH media. Drug carriers

DLC (%) pH 5.0 pH 6.5 pH 7.4

CPMAA@CPNIPAM

7.5

8.2

10.2

RS-CPMAA@CPNIPAM

10.0

12.0

35.4


14

Page 15 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Due to their higher DOX-loading capacity, the in vitro drug release from the DOX-loaded CPMAA@CPNIPAM and RS-CPMAA@CPNIPAM microspheres at pH 7.4 were conducted at different PBS media (pH 7.4, or 5.0 mimicking the normal body fluids, or the endolysosomal internal milieu19) and different temperatures (37 or 25 °C). Their cumulative release profiles as a function of soaking time are given in Figure 4. Both the DOX-loaded microspheres exhibited faster releasing rate at acidic media and the higher temperature, indicating their pH and temperature dual-responsive characteristic. However, the DOX-loaded CPMAA@CPNIPAM microspheres showed an obvious burst release at 37 °C due to the DOX molecules loaded into the CPNIPAM shells, in comparison with the DOX-loaded RS-CPMAA@CPNIPAM microspheres showing a sustained release behavior, especially at pH 5.0 media. At the releasing temperature higher than the LCST of PNIPAM (about 32 °C), the CPNIPAM shells shrank, as a result the DOX molecules loaded in the CPNIPAM shells were squeezed out quickly. For both microspheres, the CPNIPAM shells shrank to squeeze out the DOX molecules loaded into the CPMAA cores at higher temperature (37 °C), so the higher cumulative release rates were achieved. Based on an overall consideration of the lower DLCs and the burst release of the DOXloaded CPMAA@CPNIPAM microspheres, it could be concluded that the CPNIPAM shells did limit

the

volume

expansion

during

the

drug-loading.

Although

the

DOX-loaded

CPMAA@CPNIPAM microspheres presented a relatively higher cumulative release rate than the DOX-loaded RS-CPMAA@CPNIPAM microspheres, more DOX molecules had been released from the DOX-loaded RS-CPMAA@CPNIPAM microspheres because of their higher DLC. Most importantly, the DOX-loaded RS-CPMAA@CPNIPAM microspheres presented the lowest drug release rate at pH 7.4 and 37 °C, meaning they must have the minimal drug leakage during


15


the body circulation. The result demonstrated that the novel raspberry-shaped yolk/shell microspheres possessed excellent site-specific targeted release performance.

50

Cumulative Release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

40 30 o

20

pH5.0-37 oC pH7.4-37 oC pH5.0-25 C

CPMAA@CPNIPAM CPMAA@CPNIPAM CPMAA@CPNIPAM

RS-CPMAA@CPNIPAM RS-CPMAA@CPNIPAM RS-CPMAA@CPNIPAM

10 0 0

10

20

30

40

50

60

Time/h

Figure 4. Cumulative release from the DOX-loaded core/shell CPMAA@CPNIPAM and yolk/shell RS-CPMAA@CPNIPAM microspheres.

The difference in the release performance of the two drug carriers might be resulted from their different crosslinking structures, which has significant influence on their swelling behavior. Compared the swelling ratios (volume ratio of the microspheres after and before swelling in water at room temperature for 24 h, denoted as (Dh(nm)25°C/DTEM(nm))3) of the two microspheres,3 one can easily find that the value of the core/shell CPMAA@CPNIPAM (swelling ratio of 59) is much higher than that of the yolk/shell RS-CPMAA@CPNIPAM microspheres (swelling ratio of 36), although their shrinking percentages at 37 °C were similar.


16

Page 17 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Due to the higher reactivity ratio of DVB than NIPAM, more DVB would polymerize in the initial stage of polymerization.20 Thus a radial gradient in the crosslinking degree formed in the CPNIPAM shells of the regular CPMAA@CPNIPAM core/shell microspheres via the one-step seeded emulsion copolymerization, decreasing from inside to outside. As for the yolk/shell RSCPMAA@CPNIPAM microspheres, more nuclei were formed with higher crosslinking degree of DVB at the in the initial stage of polymerization.20 As a result, the CPNIPAM nanogels in the CPNIPAM shells of the yolk/shell RS-CPMAA@CPNIPAM microspheres should have the higher crosslinking degree. However, the CPNIPAM nanogels integrated with each other later, so the crosslinking degree in the linkage structure should be lower. In a word, the high-crosslinkingdegree section determines the shrinking percentage, while the swelling ratio depends on the proportion with low-crosslinking degree. Thus it could be concluded that the nonhomogeneous crosslinking structures in the two microspheres should be the most determining factor of their different releasing behaviors. To demonstrate the phenomena, the drug release mechanism from the two kinds of CPMAA@CPNIPAM microspheres was explored with the Korsmeyer-Peppas model (Figure S1). At pH 5.0 and 37 °C, both the equations yielded comparatively good linearity (most of them > 0.90). However, the exponent n = 0.3142 for the CPMAA@CPNIPAM core/shell microspheres indicates Fickian diffusion while n = 0.6145 for the RS-CPMAA@CPNIPAM yolk/shell microspheres indicates non-Fickian model (anomalous transport).11,12 The distinctive feature of their morphologies and especially the nonhomogeneous crosslinking structure, resulting from the different fabrication methods, mainly contributes to the different release kinetics in the two kinds of CPMAA@CPNIPAM microspheres.

CONCLUSIONS


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 22

In summary, novel raspberry-shaped independent temperature and pH dual-responsive RSCPMAA@CPNIPAM yolk/shell microspheres were designed as potential drug delivery system for anticancer drugs via the one-pot two-step “self-removing” approach. For the model drug DOX, the RS-CPMAA@CPNIPAM yolk/shell microspheres showed higher drug-loading capacity, better stimuli-responsive controlled release while minimal drug leakage during the body circulation, in comparison with the regular CPMAA@CPNIPAM core/shell microspheres via the one-step seeded emulsion copolymerization, due to the distinctive feature of their morphologies and nonhomogeneous crosslinking structure.

ASSOCIATED CONTENT The drug release models. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author. * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.

ACKNOWLEDGMENTS This project was granted financial support from the National Natural Science Foundation of China (Grant No. 20904017) and the Program for New Century Excellent Talents in University (Grant No. NCET-09-0441).


18

Page 19 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Cheng, R.; Meng, F.H.; Deng, C.; Klok, H.A.; Zhong, Z.Y. Dual and Multi-stimuli Responsive Polymeric Nanoparticles for Programmed Site-specific Drug Delivery. Biomaterials 2013, 34, 3647−3657. (2) Zhu, L.; Torchilin, V.P. Stimulus-responsive Nanopreparations for Tumor Targeting. Intergr. Biol. 2012, 5, 96−107. (3) Liu, L.; Liu, P. Independent Temperature and pH Dual-stimuli Responsive Yolk/shell Polymer Microspheres for Controlled Release: Structural Effect. Eur. Polym. J. 2015, 69, 540−551. (4) Li, X.R.; Du, P.C.; Liu, P. Layer-by-layer Polyelectrolyte Complex Coated Poly(methacrylic acid) Nanogels as a Drug Delivery System for Controlled Release: Structural Effects. RSC Adv. 2014, 4, 56323−56331. (5) Du, P.C.; Liu, P. Novel Smart Yolk/Shell Polymer Microspheres as a Multiply Responsive Cargo Delivery System. Langmuir 2014, 30, 3060−3068. (6) Ji, H.F.; Wang, S.P.; Yang, X.L. Preparation of Polymer/Silica/Polymer Tri-layer Hybrid Materials and the Corresponding Hollow Polymer Microspheres with Movable Cores. Polymer 2009, 50, 133−140. (7) Li, G.L.; Shi, Q.; Yuan, S.J.; Neoh, K.G.; Kang, Y.T.; Yang, X.L. Alternating Silica/Polymer Multilayer Hybrid Microspheres Templates for Double-shelled Polymer and Inorganic Hollow Microstructures. Chem. Mater. 2010, 22, 1309−1317. (8) Du, P.C.; Wang, T.M.; Liu, P. Double-walled Hollow Polymeric Microspheres with Independent pH and Temperature Dual-responsive and Magnetic-targeting Function from Onion-shaped Core–shell Structures. Colloid Surf. B-Biointerfaces 2013, 102, 1–8.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 22

(9) Qian, J.; Wu, F.P. Synthesis of Thermosensitive Hollow Spheres via a One-pot Process. Chem. Mater. 2007, 19, 5839−5841. (10)

Weda, P.; Trzebicka, B.; Dworak, A.; Tsvetanov, C.B. Thermosensitive Nanospheres of

Low-density core – An Approach to Hollow Nanoparticles. Polymer 2008, 49, 1467–1474. (11)

Pulat, M.; Asil, D. Fluconazole Release Through Semi-Interpenetrating Polymer Network

Hydrogels Based on Chitosan, Acrylic Acid, and Citraconic Acid. J. Appl. Polym. Sci. 2009, 113, 2613–2619. (12)

Raval, A.; Parikh, J.; Engineer, C. Mechanism and in Vitro Release Kinetic Study of

Sirolimus from a Biodegradable Polymeric Matrix Coated Cardiovascular Stent. Ind. Eng. Chem. Res. 2011, 50, 9539–9549. (13)

Subotic, D.V.; Wu, X.Y. Interactions of Poly(N-isopropylacrylamide-co-methacrylic

acid) with Poly(N-vinylpyrrolidone). Ind. Eng. Chem. Res. 1997, 36, 1302–1309. (14)

Zhang, K.; Wu, W.; Chen, J.; Zhang, P. Synthesis of Temperature-responsive Poly(N-

isopropyl acrylamide)/Poly(methyl methacrylate)/Silica Hybrid Capsules from Inverse Pickering Emulsion Polymerization and their Application in Controlled Drug Release. Langmuir 2010, 26, 7971−7980. (15)

Bradley, M.; Vincent, B. Poly(vinylpyridine) Core/Poly(N-isopropylacrylamide) Shell

Microgel Particles: Their Characterization and the Uptake and Release of an Anionic Surfactant. Langmuir 2008, 24, 2421−2425. (16)

Zhuk, A.; Mirza, R.; Sukhishvili, S. Multiresponsive Clay-Containing Layer-by-Layer

Films. ACS Nano 2011, 5, 8790−8799. (17)

Wang, G.Y.; Maciel, D.; Wu, Y.L.; Rodrigues, J.; Shi, X.Y.; Yuan, Y.; Liu, C.S.; Tomas,

H.; Li, Y.L. Amphiphilic Polymer-Mediated Formation of Laponite-Based Nanohybrids with


20

Page 21 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Robust Stability and pH Sensitivity for Anticancer Drug Delivery. ACS Appl. Mater. Interfaces 2014, 6, 16687−16695. (18)

Chen, L.-B.; Zhang, F.; Wang, C.-C. Rational Synthesis of Magnetic Thermosensitive

Microcontainers as Targeting Drug Carriers. Small 2009, 5, 621−628. (19)

Wojtkowiak, J. W.; Verduzco, D.; Schramm, K. J.; Gillies, R. J.Drug Resistance and

Cellular Adaptation to Tumor Acidic pH Microenvironment Mol. Pharmaceutics 2011, 8, 2032–2038. (20)

Li, G.L.; Song, Y.Y.; Yang, X.L.; Huang, W.Q. Preparation of Poly(divinylbenzene-co-

isopropylacrylamide) Microspheres and Their Hydrogen-Bonding Assembly Behavior for Raspberry-Like Core-Corona Polymer Composite. J. Appl. Polym. Sci. 2007, 104, 1350–1357.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 22

For Table of Contents Use Only

Raspberry-shaped independent temperature and pH dual-responsive CPMAA@CPNIPAM yolk/shell microspheres for site-specific targeted delivery of anti-cancer drugs Lei Liu, Jinshan Guo, Peng Liu*

APS SDS

DVB, APS and SDS

NIPAM DVB APS SDS

CPMAA@PNIPAM growing and connecting

CPMAA@CPNIPAM

washing with water at r.t.

RS-CPMAA@CPNIPAM CPMAA@PNIPAM@CPNIPAM


22

Raspberry-Shaped Independent Temperature and pH Dual

Recommend Documents