Doxorubicin-Functionalized Silica Nanoparticles Incorporated into a

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Doxorubicin-functionalized silica nanoparticles incorporated into a thermoreversible hydrogel and intraperitoneally administered result in high prostate antitumor activity and reduced cardiotoxicity of doxorubicin Camila P. Silveira, Leticia M. Apolinário, Wagner J. Favaro, Amauri Jardim Paula, and Nelson Duran ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00241 • Publication Date (Web): 08 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 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.

ACS Biomaterials Science & Engineering 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 37

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

ACS Biomaterials Science & Engineering

Doxorubicin-functionalized

silica

nanoparticles

incorporated

into

a

thermoreversible hydrogel and intraperitoneally administered result in high prostate antitumor activity and reduced cardiotoxicity of doxorubicin

Camila P. Silveira,§,# Letícia M. Apolinário,¶ Wagner J. Fávaro,¶,¥,* Amauri J. Paula,†,* Nelson Durán§,#,¥ §

Laboratory of Biological Chemistry, Institute of Chemistry, Universidade Estadual de

Campinas (UNICAMP), R. Monteiro Lobato, P.O. BOX 6154, 13083-970, CampinasSP, Brazil #

NanoBioss, Institute of Chemistry, Universidade Estadual de Campinas (UNICAMP),

R. Monteiro Lobato, P.O. BOX 6154, 13083-970, Campinas-SP, Brazil ¶

Department of Structural and Functional Biology, Institute of Biology, Universidade

Estadual de Campinas (UNICAMP), R. Monteiro Lobato, P.O. BOX 6109, 13083865, Campinas-SP, Brazil ¥

Farmabrasilis R&D Division, Campinas-SP, Brazil

†

Solid-Biological Interface Group (SolBIN), Department of Physics, Universidade

Federal do Ceará (UFC), Campus do Pici, P.O. BOX 6030, 60440-900, Fortaleza-CE, Brazil

Corresponding Authors * Tel.: +55 19 3521 6104; email: [email protected] * Tel.: +55 85 3366 9270; email: [email protected]

Keywords: mesoporous silica nanoparticles, Pluronic F-127, poloxamer, hydrogel, doxorubicin, prostate cancer, cardiotoxicity, drug delivery

1 Environment ACS Paragon Plus


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

ABSTRACT

Described here is an anticancer material based on colloidal mesoporous silica nanoparticles (MSNs) functionalized with doxorubicin (DOX), and incorporated into Pluronic F127 hydrogels for prolonged release, with a potential therapeutic application for prostate cancer treatment. The MSNs have spherical morphology, size of about 60 nm, surface area of 970 cm2 g−1 and average pore width of 2.0 nm. A high colloidal stability for the MSNs in the physiological medium used for in vivo administration (NaCl 0.9% w/v) could be attained in the presence of PF127 (from 5 to 18 wt%), where depletion repulsion forces prevent MSN agglomeration. By conjugating DOX, MSN and PF127 (18 wt%) in NaCl 0.9%, the hybrid system has a gelation temperature of 21 °C, which allowed its in vivo administration in the liquid form and further in situ gelation, generating a drug depot system inside the animals after peritoneal injection. The systems were tested in rats with chemically-induced prostate cancer and, after this treatment, histopathological analyses confirmed (i) a reduction in the frequency of aggressive tumors; (ii) that the antitumor effect was dependent on MSN concentration; and most importantly (iii) the reduction of DOX intrinsic cardiotoxicity, indicating that the MSNs play a cardioprotective effect.


Page 2 of 37

Page 3 of 37

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


INTRODUCTION

The use of nano-based materials for sustained release of drugs presents advantages over the use of free drugs. These materials can improve the availability of molecules in the body1 and, when target-specific, can increase the drug concentration in sites of interest,2,3 thus enabling the use of smaller doses. More specifically, nanomaterials emerge as a great alternative for improving chemotherapies,4 since tumor characteristics favor the target specificity of nanostructures. To date, therapeutic options for the majority of cancers involve the use of highly cytotoxic drugs, which are related to a high incidence of side effects. This aspect limits to some extent the use of these drugs and, although very efficient, oncologists must balance cost-benefits between efficiency and toxicity.5,6 In this context, mesoporous silica nanoparticles (MSNs) are attractive for their high biocompatibility and efficiency in carrying actives and biomolecules.7–9 These particles have a high surface area and large pore volume, suitable for carrying drugs.10 Furthermore, MSNs have low toxicity,11 being rapidly eliminated by the organism,9,12 which differs from the majority of inorganic materials. These features insert them into a wide range of biomedical applications, which have resulted in several investigations applying MSNs in the treatment of several types of cancer with very promising results.13–15 Another strategy to ensure better drug efficiency is the use of prolonged release devices, such as drug depot systems.16 These systems are injected into the organism and form a depot in situ, releasing their components gradually, and thus increasing the drug residence time.17 An excellent alternative to achieve these features is the association of drugs to thermoreversible polymers,18,19 in which a solution undergoes



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

gelation under certain temperature ranges. Among these, Pluronic F-127 (PF127), also known as poloxamer 407, is a thermoreversible block copolymer, biocompatible20 and FDA-approved, that can present a liquid-gel transition temperature near room temperature.21 That means the polymer solution undergoes gelation when the temperature increases to room temperature, thus the drug dissolved in the polymeric solution can be injected into the patient, gelling in situ and resulting in a drug depot for prolonged release. By conjugating both features, described here is the production of a drug depot system based on a PF127 hydrogel, MSNs and doxorubicin (DOX), which resulted in a platform for anticancer applications. DOX is an antitumor drug of large spectrum that causes a high incidence of cardiotoxic effects and drug resistance. For this reason, there is a great interest in reducing DOX side effects without altering its antitumor activity.22 The combination of MSNs+DOX+PF127 towards the production of a multi-component hybrid system was optimized based on a study of the physicochemical interactions of MSNs in combination with both DOX and PF127, and the efficiency of the hybrid system produced was tested in rats with chemicallyinduced prostate cancer. The results encourage the use of hybrid systems as a platform for cancer treatment and open a perspective towards new prostate cancer therapies.

EXPERIMENTAL

Synthesis and characterization of MSNs MSNs were synthesized following the protocol developed by Paula et al.:23 a sol-gel method based on the concepts and methods proposed by Stöber24 and Bein.25


Page 4 of 37

Page 5 of 37

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 synthetic approach used allows the production of spherical MSNs that are highly stable in aqueous medium, with an average size of about 60 nm (with a very narrow size distribution of 45–75 nm), high surface area (~1,000 m2 g−1), inner pores of ~2 nm diameter and a large pore volume (around 1.0 cm3 g−1). For the MSN production, 0.75 g of cetyl trimethyl ammonium bromide (CTAB, obtained from Sigma-Aldrich) was dissolved in 20 mL of a 0.050 mol L−1 ammonium hydroxide (NH4OH, 28% v/v) solution (pH 11.1) and homogenized in an ultrasound bath for 10 min. To this solution, 3.2 mL of absolute ethanol was added and, after 15 minutes under magnetic stirring, 2.5 mL of tetraethyl orthosilicate (TEOS, obtained from Acros Organics) was added. The reaction was kept under reflux at 60 °C for 2 h and then the products were separated by centrifugation at ~18,000 rcf for 60 min (Beckman Coulter™ Allegra™ X-22R Centrifuge). To accomplish the CTAB extraction, absolute ethanol was added so that the volume reached ~90 mL and the suspension was sonicated for 5 min. Then, 10 mL of hydrochloric acid (HCl, analytical purity, obtained from LabSynth) was added and the suspension was sonicated for 10 min. Nanoparticles were finally obtained by centrifuging the suspension for 60 min and washing twice with absolute ethanol. MSNs were characterized with regard to their morphology by transmission electron microscopy (TEM) in bright field mode (TEM-BF, Zeiss Libra 120, operating at 80 kV). Zeta (ζ) potential and particle size analyses were carried out at a 0.250 mg mL−1 nanoparticle concentration in 10×-diluted PBS and in deionized water, respectively. Size and ζ-potential values were evaluated by dynamic light scattering analysis (DLS, Malvern ZetaSizer-Nano ZS). The nanoparticle surface area, pore volume and pore diameter were obtained through nitrogen-sorption experiments (Accelerated Surface Area and Porosimetry System, ASAP 2020, Micromeritics). The



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 37

Brunauer–Emmett–Teller (BET) method was used to calculate surface area and the Barrett–Joyner–Halenda (BJH) method to calculate the pore diameter, both through the N2 adsorption branch. Pore volume was calculated from the single-point value adsorbed at P/P0 = ~0.94.

MSN interactions with DOX To evaluate the DOX loading capacity of MSNs and their surface charge distribution in the presence of DOX, MSNs were firstly incubated with different DOX concentrations. The ζ-potential of the MSNs in each sample was measured and the samples were subsequently centrifuged to pelletize DOX–MSN complexes. The quantification of DOX loaded into MSNs was carried out by calculating the amount of free DOX in the supernatant by measuring its light absorbance (Micronal AJX6100PC) at 480 nm (see Supporting Information B1). DOX loading efficiency was calculated according to Equation 1, and the loading capacity of MSNs was calculated according to Equation 2.

=

"#$ =

100

100

Equation 1

Equation 2

Colloidal stability of MSNs The effect of PF127 on the colloidal stability of the nanoparticles was assessed through a centrifugation study. MSNs were incubated with different concentrations of PF127 in the presence of NaCl 0.9% (w/v) and the samples centrifuged at different relative centrifugal forces (90; 2,340; 9,340 and 18,400 rcf) for 5 min. The amount of MSNs left in the supernatant was quantified by inductively coupled plasma optical 6 Environment ACS Paragon Plus

Page 7 of 37

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


emission spectrometry (ICP-OES, PerkinElmer Optima 8300, see Supporting Information A2) by means of silicon. To evaluate the aggregation profile of the MSNs in different PF127 concentrations, the average particle size and polydispersity index were measured as a function of time through DLS.

Synthesis and characterization of hydrogel hybrid systems Preparation of the PF127 (30 wt%) solution: the concentrated PF127 solution was prepared on a weight basis using the cold method.26 A proper amount of PF127 to yield 30 wt% was added to physiological saline solution (NaCl 0.9% w/v) at 4 °C and kept under vigorous magnetic stirring overnight until the formation of a clear solution. Preparation of the hybrid systems: the hybrid systems were synthesized by mixing the exact mass of DOX to result in a 1.25 mg mL−1 solution (see Supporting Information A3) and PF127 30 wt% in physiological saline solution, in order to generate a PF127 18 wt% solution. MSNs were added last to avoid aggregation. The final mixture was kept under magnetic stirring and in a cold bath overnight. Gelation temperature (Tgel): the Tgel assay was accomplished by transferring 5 mL of the PF127 solution (18 wt%) to a beaker in a cold bath with a thermometer. The solution was heated under a constant rate and magnetic stirring. Tgel was considered the temperature at which the magnetic bar stopped moving. Gel dissolution and drug release: the in vitro assay to evaluate the gel dissolution rate and drug release was accomplished through a membraneless dissolution method in a dry bath at 35 °C (corresponding to the body temperature of Fischer 344 rats used in the in vivo assay). In this method, the release medium is introduced carefully onto the surface of the gel and the dissolution rate is calculated through the gel weight loss over time, by repeatedly changing the release medium to



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

avoid saturation of the solution, such as occurs in biological fluids, in which there is a constant concentration gradient. For this, 500 µL of the hydrogel was transferred to a weighted vial and incubated in a dry bath at 35 °C to reach thermal equilibrium. Then, 500 µL of the release medium (1 mg mL−1 of BSA solution, simulating a release medium containing biomolecules) was poured carefully onto the surface of the hydrogel (see Supporting Information A3, Figure S2). At pre-determined intervals, the release medium was removed and the vial weighed. A new release medium was then added to avoid saturation. To calculate the amount of DOX released, each release medium was centrifuged to pellet the DOX–MSN complex (with DOX still incorporated into MSNs), and finally the amount of drug released was calculated by measuring the light absorbance of the supernatant at 480 nm.

In vivo assays Seventeen male rats (7-week-old Fischer 344) were used in this study. They were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at the University of Campinas (UNICAMP). Four of them comprised the Control group (healthy animals, untreated, n = 4) and prostate cancer induction was performed in the remaining 13 animals according to a new protocol (see Supporting Information A4) that enables the animals to be ready for treatment in 120 days. After this period, the 13 rats with prostate cancer were divided into four groups: Cancer Control (untreated, n = 4), Control-DOX (n = 3), G1-DOX (n = 3) and G2-DOX (n = 3). Each group received a weekly dose of 0.3 mL of the respective treatment intraperitoneally for 30 days (totalizing four applications). To prevent gelation of the systems inside the syringe and needles and consequent loss of product, the systems were kept in an ice bath before each application. This allowed a better flow of the


Page 8 of 37

Page 9 of 37

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


system through the needle and gelation only inside the animal body. The animals received water and the same solid diet ad libitum (Nuvilab) and were allocated to single solid-bottom boxes lined with wood shavings in a room with controlled light and temperature (12 h light and 12 h dark, 20‒25 °C). The experimental protocol followed ethical principles in animal research (CEUA/UNICAMP – Protocol no 30561).

Histopathology After one week from the last dose application, the animals were euthanized and samples of prostatic ventral lobe and transverse sections of the heart of each animal underwent histopathological analysis. The samples were collected and fixed in Bouin solution for 12 h. After fixing, the tissues were washed with ethanol 70% (v/v) and dehydrated. In addition, the fragments were diaphonized in xylol and embedded in a plastic polymer (Paraplast Plus, St. Louis, MO, USA). The fragments were cut to attain 5 µm thickness by using a microtome Biocut 1130 (Reichert-Jung), stained with hematoxylin-eosin and photographed using a Nikon Eclipse Ni-U photomicroscope (equipped with a Nikon DS-RI-1 camera). The diagnoses of both prostatic and heart lesions were based on morphological features.

RESULTS

Characterization of bare MSNs MSNs were prepared through a sol-gel method based on the Stöber protocol,23,24 which uses silicon alkoxides as precursors. After the hydrolysis and condensation reactions in an ammoniacal medium, a new sol phase was formed after the nucleation



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

and growth of the colloidal particles.27 Absolute ethanol was used as a homogenizing agent, whereas a cationic surfactant was used as the template to generate the pores. This method was able to produce porous nanoparticles with high colloidal stability.28 TEM images (Figure 1) revealed nanoparticles with spherical morphology with a size distribution of 45 to 75 nm. Nitrogen-sorption experiments using the BET and BJH methods indicated that the MSN surface area was 970 cm2 g−1, pore volume was 1.6 cm3 g−1 and pore diameter was 2.0 nm (see Supporting Information B2, Figure S4). The adsorption-desorption isotherm had similarities with a type IV isotherm, typical of mesoporous materials, in which the initial adsorption is followed by a capillary condensation.29 The occurrence of hysteresis loops is also typical of type IV isotherms and, in the case of Figure S4b, it resembles an H1 hysteresis occurring from P/P0 = 0.8, which is associated with the presence of uniform mesopores.29 The MSN hydrodynamic radius was 117.8 ± 4.6 nm whereas the polydispersity index was 0.16 ± 0.05 and ζ-potential in 10× diluted PBS was −20.0 ± 1.0 mV.


Page 10 of 37

Page 11 of 37

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. (a) Transmission electron microscopy (TEM) images of MSNs (inset: size distribution histogram of nanoparticles obtained counting at least 100 nanoparticles on images). MSN interactions with DOX To obtain information on the DOX loading capacity of the MSNs and the surface charge profile of the MSNs in the presence of DOX, the nanoparticles (250 µg mL−1) were incubated with different DOX concentrations, resulting in different DOX : MSN (w : w) ratios. After measuring the ζ-potential of each sample, it was centrifuged to a pellet corresponding to the MSN–DOX complex, and then the DOX left in the supernatant was measured. Table 1 summarizes the results of the loading efficiency expressed as a percentage of loaded DOX as a function of total DOX. It can be observed that for the smaller DOX : MSN ratios (w : w), there was a higher percentage of DOX loaded as a function of total DOX, and as the DOX : MSN ratio increased, the percentage of DOX loaded decreased. In addition, the loading capacity (which is associated with the amount of DOX loaded per nanoparticle) increased as



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 DOX : MSN ratio increased (Figure 2). Analyzing the MSN surface charge and comparing it with the amount of DOX loaded per particle (Figure 2), it was indeed expected that the ζ-potential would be more positive as the DOX amount was increased, since DOX pKa is 9.2 and there is an amount of the molecule adsorbed on the external surface of the MSNs.

Table 1. DOX loading efficiency for MSNs evaluated by keeping the nanoparticle concentration constant. DOX : MSN

Loading

ratio (w : w)

efficiency* (%)

0.25

63.20 ± 1.60

0.50

54.79 ± 0.45

0.75

45.17 ± 1.35

1.00

34.74 ± 1.34

1.25

35.02 ± 5.40

* Standard deviation was calculated considering at least three independent measurements.


Page 12 of 37

Page 13 of 37

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 2. MSN ζ-potential profile (blue curve) and the amount of DOX loaded per MSN mass unit (red bars) as a function of DOX : MSN ratio. Black bars in the graphics represent standard deviations calculated from at least three measurements for independent samples.

Colloidal stability of MSNs in a PF127 solution: evidence of short- and longrange interactions To check whether PF127 would lead to a stabilizing effect on the MSN suspension, the colloidal stability of MSNs was studied in a wide range of PF127 concentrations (0.1 to 6.0 wt%) through centrifugation studies, and ζ-potential and particle size assays. It should be mentioned that there are some limitations of these assays due to the thermosensitive nature of PF127, which limits the centrifugation studies to PF127 concentration ranges that do not result in hydrogel formation at room temperature (up to 10 wt%). In addition, it is known that PF127 is a depletant agent28 that can induce stabilization or destabilization depending on the concentration range. Therefore ζ-potential measurements could only be performed in PF127 concentration ranges in which colloid stability was maintained. Firstly, we evaluated the stability of MSNs in the presence of PF127 in regard to their aggregation rate as a function of time, monitoring average particle size and polydispersity index (through DLS) at low PF127 concentrations (from 0.01 to 0.5 wt%). The results indicated that at



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

concentrations lower than 0.1 wt% (Figure 3, left), there was an increase in the particle size that was immediately observed in the first minute of the test, indicating agglomeration of the colloid (colloidal instability). It can also be noted that the colloidal stability was achieved as the PF127 concentration was increased, suggesting that depletion forces rule the stabilization process. In this type of interaction, entropic forces originate from fluctuations in the osmotic pressure throughout the sample and from the excluded volume effect determine whether the particles would experience stabilization or destabilization. In this mechanism of colloidal stabilization, MSNs can be seen as large particles that are prevented from agglomeration at certain concentrations of small particles present in the suspension (i.e., PF127 micelles; a deeper discussion on colloidal stabilization through repulsive depletion forces is found in Supporting Information A1). At concentrations of PF127 above 0.1 wt% (Figure 3, right), agglomeration was not observed within the time range of the test (60 min), although there was a slight increase in the average particle size in comparison with bare MSNs (red curve). This increase was not followed by an increase in the polydispersity index, as would be expected if aggregation had occurred. Therefore, it can be related to the formation of a layer of PF127 around the particles, which was confirmed by the decreasing values of ζ-potential for the MSNs in the presence of increasing concentrations of PF127 (see Supporting Information B3, Figure S5).


Page 14 of 37

Page 15 of 37

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 3. Aggregation profile of the MSNs (250 µg mL−1) at different PF127 concentrations (in wt%) measured through dynamic light scattering (DLS) with dispersion in deionized water. Hydrodynamic radii from 0 to 800 nm are expanded in the graphic on the left. PDI: polydispersity index. Bars in the graphics represent standard deviations calculated from at least three measurements for independent samples.

Taking into account that the hybrid systems would be produced in physiological saline solution (NaCl 0.9% w/v), we accomplished a centrifugation study in this conditions. Firstly, we studied the colloidal stability of MSNs covering a range of PF127 concentrations from 0.1 to 5.0 wt%, and using 500 µg mL−1 of MSNs (Figure 4a). The manifestation of depletion forces for the mixture of MSNs and PF127 was confirmed in deionized water, in which a concentration of 0.1 wt% was able to stabilize the MSN suspension, whereas smaller concentrations led to agglomeration of the colloid (Figure 3). In NaCl 0.9% w/v, the attractive depletion forces become evident as PF127 was added at low concentrations, causing a sharp destabilization, which was coherent with the previous tests. As PF127 concentration increased (5.0 wt%), stability increased gradually. Using the same suspensions from the test of



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 4a, photographs of the samples were taken over 8 days (see Supporting Information, Figure S1e) to verify whether it would be possible to visually identify aggregation. A clear phase separation (colloidal destabilization) could be observed due to MSN aggregation in sample 2 (0.1 wt% of PF127). As PF127 concentration increased, the suspension was stabilized. It should also be taken into account that the increase in PF127 concentration increased the system viscosity, and this aspect is also important in the results observed. By doubling the MSN concentration to 1.0 mg mL−1 in NaCl 0.9% w/v and in PF127 concentrations from 3.0 to 6.0 wt%, another centrifugation study was performed (Figure 4b). Higher PF127 concentrations were not studied because of hydrogel formation. These results also showed high stability of the MSNs in physiological saline solution, and that PF127 was performing stabilization when at high concentrations. By increasing PF127 concentration, the amount of MSNs that remained in the supernatant when the sample was submitted to the highest rcf (18,000 rcf) was increased, indicating stabilization. Therefore, it has been observed that PF127 acts as an initial stabilizing agent for MSNs until the moment of administration in the animals.


Page 16 of 37

Page 17 of 37

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 4. (a) Centrifugation study for assessing MSN (500 µg mL−1) colloidal stability in NaCl 0.9% (w/v) and PF127 concentrations from 0.1 to 5.0 wt%, analyzed through ICP-OES after centrifuging the samples for 5 min. (b) Colloidal stability of MSNs (1.0 mg mL−1) in NaCl 0.9% (w/v) and PF127 concentrations from 3.0 to 6.0 wt%, analyzed through ICP-OES after centrifuging the samples for 5 min. Bars in the graphics represent standard deviations calculated from at least three measurements for independent samples.

Hybrid system preparation and characterization Aiming

to

evaluate

the

antitumor

activity

of

the

association

hydrogel+MSNs+DOX and the role of MSNs in this process, two formulations (namely hybrid systems) were prepared. The only difference between the systems was



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 37

the MSN concentration. The systems (Table 2) were named G1-DOX (with a lower MSN concentration), G2-DOX (with a higher MSN concentration) and the control system was named Control-DOX. All systems were characterized regarding their gelation temperature, which lay at around 20 °C, being considered a suitable temperature range in order to ensure easier manipulation of the hydrogel solution prior to in vivo administration (see Supporting Information B4).

Table 2. Detailed description of the hybrid systems produced System

Components PF127

Control-DOX

G1-DOX

G2-DOX

Final concentration 18.0 wt%

Tgel* (°C) 21.0 ± 0.5

−1

DOX

1.25 mg mL

PF127

18.0 wt%

DOX

1.25 mg mL−1

MSNs

1.0 mg mL−1

PF127

18.0 wt%

DOX

1.25 mg mL−1

MSNs

5.0 mg mL−1

21.0 ± 0.5

22.0 ± 0.50

* Standard deviation was calculated considering at least three independent measurements.

Gel dissolution and drug release The hydrogel dissolution and drug release assays were performed to confirm the prolonged release of DOX by comparing the gel dissolution and drug release curves. Considering the gel dissolution and drug release profile for Control-DOX (without nanoparticles), drug release from the hydrogel in contact with the release medium occurs due to two concurrent processes related to drug flux and water flux. The first


Page 19 of 37

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


process is drug diffusion through the release medium, governed by a chemical potential. The second is hydrogel dissolution by the water present in the release medium, prior to thermodynamic equilibrium, thus releasing the drug. It is assumed that drug diffusion (governed by Fick’s law) controls release during the first moments of the assay, until the hydrogel starts to dissolve.28 From this moment, hydrogel dissolution rules drug release.28 It can be noted that the hydrogel dissolution and drug releases profile for Control-DOX (Figure 5a) are not an asymptotic function, which would be expected for a releasing system evolving towards equilibrium. This difference is explained by the fact that the release medium was removed at pre-determined intervals and a new release medium was added, altering the thermodynamic conditions of the process, exactly as occurs in vivo. Therefore, this protocol analyzed the release profile of a system that does not reach equilibrium, a situation analogous to the biological medium in which the system would be introduced, where cells (and consequently tissues) induce a constant concentration gradient in the liquid. In the case of hydrogels containing nanoparticles, the complexity increases once other processes occur during drug release. Nanoparticle diffusion throughout the hydrogel and sorption equilibrium between DOX and MSNs must be considered in both the hydrogel and release medium. So, to confirm whether MSNs were capable of promoting a prolonged release of DOX, the hydrogel dissolution and drug release curves were compared for G1-DOX and G2-DOX systems (Figure 5b and c). In the dissolution curve, data was drawn with nanoparticles and DOX present in the release medium, while the drug release curves were obtained by measuring just DOX in the release medium (supernatant), since the MSNs were centrifuged. The difference between the two curves (dissolution and release) indicates that part of DOX that can diffuse to the



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

release medium is still incorporated in the MSNs, thus indicating that a prolonged release does indeed occur. Particularly, for the G2-DOX system, there was a larger difference between the two curves, possibly because of the higher MSN concentration. This difference was smaller for G1-DOX, which could be associated with the lower MSN concentration, resulting in a prolonged release that was not so pronounced for the time interval measured (30 min).

In vivo assays To evaluate the antitumor activity of the systems developed, the animals were divided into five groups. Each group received weekly intraperitoneal application of the respective treatment for 30 days (Table 3). The formulations were kept in an ice bath before each application to avoid loss of samples due to gelation inside the syringe needles (Figure 6).


Page 20 of 37

Page 21 of 37

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 5. (a) Gel disruption patterns of PF127 hydrogel (Control) and of PF127 hydrogels containing MSNs at different concentrations: 1.0 mg mL−1 (G1-DOX) and 5.0 mg mL−1 (G2-DOX). The hydrogel disruption (in % of its initial weight) was calculated by gravimetry assays performed after a progressive exchange of the supernatant liquid medium. Each point in the graph corresponds to an individual medium exchange. (b) DOX release from the same systems (Control, G1-DOX and G2-DOX) assessed after exchange of the supernatant liquid medium, as described for (a). The DOX released (in % of its initial amount incorporated in the hydrogel: 1.25 mg mL−1) towards the supernatant liquid medium was calculated after progressive exchange of the liquid medium, and the DOX amount was calculated through UV-Vis absorption spectroscopy. Bars in the graphics represent standard deviations calculated from at least three measurements for independent samples.



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 6. Schematic representation of application of the systems in the animals with in situ gelation.

Table 3. Brief description of the in vivo applications Group

Application

Control (healthy rats)

5 mL kg−1 physiological saline 0.9% (w/v)

Cancer Control (rats with cancer)

5 mL kg−1 physiological saline 0.9% (w/v)

Control-DOX

System Control-DOX

G1-DOX

System G1-DOX

G2-DOX

System G2-DOX


Page 22 of 37

Page 23 of 37

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


After treatment, histopathological analysis of the prostatic tissue (Figure 7) enabled evaluation of the antitumor potential of the systems (see Supporting Information A4 for more information), which was confirmed for both G1-DOX and G2-DOX. The quantitative results shown in Table 4 result from observation of the presence of the elements described in Figure 7. There was a reduction in the frequency of more aggressive tumors (high and intermediate grade), which gave place to a higher incidence of low-grade tumors (Table 4). It can be seen that the improvement in tumor conditions was proportional to the MSN concentration in the systems, suggesting a determinant role of the MSNs in this process.



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 7. Histopathological analysis. (a) Normal epithelium with basal cell (Ep), luminal cell (Lc), lumen (L) and stroma (St). (b) Benign prostatic hyperplasia. (c) High-grade prostatic intraepithelial neoplasia (HGPIN). (d) Proliferative inflammatory atrophy (PIA) with inflammatory infiltrate (*). (e) Low-grade tumor with neoplastic acinus (Na). (f) Intermediate-grade tumor. (g) High-grade tumor with cells arranged in nests (*).


Page 24 of 37

Page 25 of 37

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


Table 4. Percentage* occurrence of histological lesions in the prostatic ventral lobe of the five experimental groups. Groups Histopathology

Normal Prostatic atrophy

Control

Cancer

Control-

G1-

G2-

(n = 4)

Control

DOX

DOX

DOX

(n = 4)

(n = 3)

(n = 3)

(n = 3)

100%

–

–

–

–

–

–

–

–

–

–

25%

–

–

–

–

75%

–

66%

100%

–

25%

–

33%

100%

–

50%

100%

66%

–

–

25%

–

–

–

Prostatic nodular hyperplasia (PNH) High-grade prostatic intraepithelial neoplasia (HGPIN) Low-grade prostatic adenocarcinoma Intermediate-grade prostatic adenocarcinoma High-grade prostatic adenocarcinoma *The percentage here represents the number of animals that presented the respective condition related to the total number of animals in the group. For example, if n = 4, 25% means one animal presented the condition and 100% means all animals presented the condition.



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

To verify whether MSNs would have any effect on DOX cardiotoxicity, the morphological heart changes of the animals were evaluated through histopathology. The diagnosis was based on cardiomyocyte degeneration, for which nuclei must be peripheral in normal conditions. Cells with central nuclei identify degeneration. From the heart histopathology results (Figure 8), a clear decrease in toxicity for the groups treated with G1-DOX and G2-DOX can be seen when compared with Control-DOX (Table 5). Even for the lower MSN concentration (1 mg mL−1, group G1-DOX), there was a significant reduction in inflammatory processes. By increasing the MSN concentration (5 mg mL−1, group G2-DOX), there was a slight improvement in the heart condition, evidenced by the decrease in inflammatory infiltration. The results strongly indicate that MSNs promote a cardioprotective effect by reducing DOX cardiotoxicity.


Page 26 of 37

Page 27 of 37

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 8. (a) Heart cross section from Control-DOX group showing intense inflammatory infiltrate (circulated area), cardiac cells (*) and their nuclei (arrow). (b) Heart cross section from G1-DOX group showing moderate inflammatory infiltrate (circled area) through transversally cut cardiac fibers (*) and their nuclei (arrow). (c) Heart cross section from G2-DOX group showing discrete inflammatory infiltrate (circled areas), cardiac fibers (*) and their nuclei (arrow). 27 Environment ACS Paragon Plus


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 28 of 37

Table 5. Percentage* occurrence of histological lesions in heart cross sections of animals treated with DOX-based systems. Grade Group ControlDOX (n = 4) G1-DOX (n = 3)

G2-DOX (n = 3)

Lesion

Slight

Moderate

Intense

Degeneration

–

66%

33%

Inflammation

–

66%

33%

Degeneration

100%

–

–

Inflammation

66%

33%

–

Degeneration

100%

–

–

Inflammation

100%

–

–

*The percentage here represents the number of animals that presented the respective condition related to the total number of animals in the group. For example, if n = 4, 25% means one animal presented the condition and 100% means all animals presented the condition.

DISCUSSION

DOX is a molecule that belongs to the class of anthracyclines that has a hydrophilic amino-sugar portion (positively charged at neutral pH) and a hydrophobic aglyconic portion. Once MSNs are negatively charged at neutral pH, as silanol groups on the silica surface tend to deprotonate towards negative charging, DOX adsorption on the MSN surface is expected to occur via electrostatic interactions, which leads to a high loading efficiency (Table 1). However, from Figure 2 it is clearly evidenced that it takes a large amount of DOX for the ζ-potential of MSNs to turn positive, which might be due to the presence of inner pores in the nanostructure. This fact indicates that the nanoparticles experience two different sorption equilibriums, one 28 Environment ACS Paragon Plus

Page 29 of 37

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


involving the MSN internal pores and the other involving the outer surface. The results also indicate that there is an adsorption preference of the drug over the internal pores of the MSNs, making this material very suitable for a drug delivery platform. To achieve good efficacy in drug delivery, it is crucial to ensure the colloidal stability of the nanoparticles, so they can act as individualized nanocarriers, preventing the occurrence of aggregation until they reach the desired site of effect. Considering a suspension comprising two species of particles (large and small), the attraction or repulsion of the large particles is related to the entropic forces originated from the osmotic pressure and from the excluded volume effect.29 These forces are called depletion forces and were first studied by Asakura and Oosawa, who developed a model that describes the depletion force acting on large particles in a solution of low concentrations of small particles.30 Around each particle there is a volume, called the depletion zone, into which the centers of the small particles cannot penetrate (excluded volume effect). When two large particles approach each other in such a way that their depletion zones overlap (i.e. the excluded volume is reduced) and no small particle can occupy the space between them, the difference between the local osmotic pressure and the bulk osmotic pressure results in an attractive force. However, at high concentrations of small particles, the correlation between them is so significant that they can interfere in the interactions and include a repulsive component,31 as shown by the Ornstein–Zernike30 theory, which considers that the depletion potential is related to the local density throughout the sample as a function of the direct correlation between particles as well as all the indirect correlations in the system (see Supporting Information A1 for a deeper discussion). From Figure 4, it is clear that the colloidal stability of MSNs depends on PF127 concentration, possibly indicating that depletion stabilization occurs. In this case, the MSNs can be associated



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

to the large particles and PF127 micelles to the small particles (some studies suggest PF127 micelles have diameter around 10 nm32). Therefore, for the systems developed in this paper, MSNs are stabilized by high PF127 concentrations, ensuring their colloidal state until administration in the animals and consequent interaction with proteins from the body fluids. Although anthracyclines such as DOX are established as one of the most efficient antitumor classes of drug, their use is still limited because of their high toxicity.6 It has been reported that DOX toxicity is associated with the production of free radicals that induce peroxidation of cardiomyocyte membranes.5 DOX intrinsic toxicity can cause permanent cardiac damage that is cumulative and dose-dependent, occurring from first contact with the drug.6 The high incidence of serious heart problems arising from the use of DOX leads oncologists to balance the possible benefits of the treatment and the cardiotoxicity risks, which can lead to heart failure.5,6 The lack of specificity of free DOX for tumor tissues causes a non-specific drug distribution, i.e. there is a considerable cellular uptake by normal tissues, including those in the heart. In this context, nanoparticles are a promising alternative in an attempt to reduce DOX toxicity. The reduction of the DOX cardiotoxicity has been reported in studies that associate this drug to nanostructures, specially liposomes.33–36,22 A known mechanism of cardiotoxicity reduction for DOX loaded in liposomal nanoparticles involves (i) the inhibition of the liposome-DOX diffusion to myocardiocytes due to the tight junctions of myocardium capillaries.37 In addition (ii) the absence of drug leakage along the liposome circulation prevents the DOX diffusion from the plasma to the myocardium, thus preventing damage to cardiac muscle cells.38 Finally, (iii) DOX that can reach the


Page 30 of 37

Page 31 of 37

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


heart is encapsulated, and can be also drained by the lymphatic system without being released, thus decreasing its toxicity.38,5 In regard to the cardiotoxicity reduction observed here when DOX was conjugated to the hybrid system (PF127+MSNs), besides the mentioned mechanisms confirmed for liposomes, there is (iv) an increase of the DOX uptake in the prostatic tumor by MSNs, which decreases the amount of DOX circulating in the organism. Insights on the mechanisms of action of sub-hundred nanometric silica nanoparticles indicate an initial accumulation in tumors by the EPR effect (i.e. enhanced permeability and retention), along with their circulation in the organism.39–41,9 Furthermore, we have recently confirmed that these MSNs accumulate in the prostatic tissue when administered by intraperitoneal injection.42 For MSNs with similar sizes of those used here, which were intravenously administered in mice, the amount of nanoparticles that reach the heart is very low in comparison to lung, liver and spleen.40,9 Another similar result was found for rigidlike silica nanoparticles of about 25 nm (diameter) intraperitoneally administered in mice.43 Furthermore, this low accumulation in the heart was observed for other nanostructures with different composition and morphology.44–46 In this way, evidences indicate that mechanisms i, ii and iii could occur to a lesser extent for the hybrid system tested here. However, more specialized analytical approaches assessing the DOX biodistribution in the presence and absence of MSNs will clarify these aspects, and other possible protective effects induced by the use of the hybrid system. The existence of other systemic effects cannot be discarded as well, mainly considering that there is the MSNs dissolution in media of high ionic strength present in the body, which also influences on the liberation of DOX molecules during their bio-circulation.47,48



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 32 of 37

CONCLUSIONS

In summary, we present here a new platform with potential therapeutic applications based on DOX-functionalized MSNs incorporated into PF127 hydrogel for prolonged release of MSNs and DOX. The system was produced from a bottomup approach which considered (i) the molecular vehicle characteristic of MSNs for incorporating and transporting DOX through the organism; (ii) the colloidal stabilizing capacity of PF127 for preventing the agglomeration of MSN sols through to the manifestation of repulsive depletion forces; and (iii) the PF127 liquid-gel thermoreversibility for gelating the hybrid system when it attains the animal body temperature after its administration in vivo as a liquid. On the colloidal stability of the MSNs, it was possible to observe that the depletant effect of PF127, which provided MSN stability in the physiological medium (NaCl 0.9% w/v), largely depends on PF127 concentration and little on that of MSNs. By increasing the PF127 concentration in the system up to that capable of generating a hydrogel, depletion repulsion forces dominate the stabilization process by preventing

nanoparticles

from

excessive

approximation

and

consequent

agglomeration. After the conjugation of DOX, MSNs and PF127, the resulting hybrid system could be handled in the liquid state up to its administration in rats, since the gelation temperature of the system was set to be around 20 °C. The hybrid system gelification resulted in a drug depot system inside the animal body, ensuring a prolonged release of DOX-functionalized MSNs. Their efficiency against prostate cancer was confirmed through an in vivo study, which indicated a substantial reduction of prostatic tumors along with a large reduction of DOX cardiotoxicity. Finally, it was observed that both


Page 33 of 37

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 antitumor effect and reduction of DOX cardiotoxicity were related to the MSN presence, opening the perspective to use these systems as a platform for drug delivery in cancer treatments.

ACKNOWLEDGEMENTS Supported by FAPESP, CNPq, FUNCAP, INOMAT (MCTI/CNPq), NanoBioSimes (MCTI/CNPq), Brazilian Network on Nanotoxicology (MCTI/CNPq) and NanoBioss (SisNano/MCTI).

SUPPORTING INFORMATION Detailed synthesis methods and experiment protocols, nitrogen-sorption experiments, physicochemical assays and also a discussion on colloidal stabilization by depletion forces are in the Supporting Information file. This material is available free of charge via the internet at http://pubs.acs.org.

REFERENCES (1)

Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Nanoparticles in Medicine: Therapeutic Applications and Developments. Clin. Pharmacol. Ther. 2008, 83, 761–769.

(2)

Allen, T. M.; Cullis, P. R. Drug Delivery Systems: Entering the Mainstream. Science 2004, 303, 1818–1822.

(3)

Qu, Q.; Ma, X.; Zhao, Y. Targeted Delivery of Doxorubicin to Mitochondria Using Mesoporous Silica Nanoparticle. Nanoscale 2015, 7, 16677–16686.

(4)

Zhang, W.; Zheng, X.; Shen, S.; Wang, X. Doxorubicin-Loaded Magnetic Nanoparticle Clusters for Chemo- Photothermal Treatment of the Prostate Cancer Cell Line PC3. Biochem. Biophys. Res. Commun. 2015, 466, 278–282.

(5)

Rahman, A. M.; Yusuf, S. W.; Ewer, M. S. Anthracycline-Induced Cardiotoxicity and the Cardiac-Sparing Effect of Liposomal Formulation. Int. J. Nanomed. 2007, 2, 567–583.

(6)

Ewer, M. S.; Ewer, S. M. Cardiotoxicity of Anticancer Treatments: What the Cardiologist Needs to Know. Nature 2010, 7, 564–575.

(7)

Li, X.; Chen, Y.; Wang, M.; Ma, Y.; Xia, W.; Gu, H. A Mesoporous Silica 33 Environment ACS Paragon Plus


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

Nanoparticle-PEI-Fusogenic Peptide System for siRNA Delivery in Cancer Therapy. Biomaterials 2013, 34, 1391–1401. (8)

Zhang, Q.; Liu, F.; Nguyen, K. T.; Ma, X.; Wang, X.; Xing, B.; Zhao, Y. Multifunctional Mesoporous Silica Nanoparticles for Cancer-Targeted and Controlled Drug Delivery. Adv. Funct. Mater. 2012, 22, 5144–5156.

(9)

Lu, J.; Liong, M.; Li, Z. X.; Zink, J. I.; Tamanoi, F. Biocompatibility, Biodistribution, and Drug-Delivery Efficiency of Mesoporous Silica Nanoparticles for Cancer Therapy in Animals. Small 2010, 6, 1794–1805.

(10)

Wu, S.; Hung, Y.; Mou, C. Mesoporous Silica Nanoparticles as Nanocarriers. Chem. Commun. 2011, 47, 9972–9985.

(11)

Lin, Y.; Haynes, C. L. Synthesis and Characterization of Biocompatible and Size-Tunable Multifunctional Porous Silica Nanoparticles. Chem. Mater. 2009, 21, 3979–3986.

(12)

He, X.; Nie, H.; Wang, K.; Tan, W.; Wu, X.; Zhang, P. In Vivo Study of Biodistribution and Urinary Excretion of Surface-Modified Silica Nanoparticles. Anal. Chem. 2008, 80, 9597–9603.

(13)

Wang, H.; Wang, F.; Zhu, X.; Yan, Y.; Yu, X.; Jiang, P.; Xing, X. Biosynthesis and Characterization of Violacein, Deoxyviolacein and Oxyviolacein in Heterologous Host, and Their Antimicrobial Activities. Biochem. Eng. J. 2012, 67, 148–155.

(14)

Ma, D.; Lin, J.; Chen, Y.; Xue, W.; Zhang, L. M. In Situ Gelation and Sustained Release of an Antitumor Drug by Graphene Oxide Nanosheets. Carbon 2012, 50, 3001–3007.

(15)

Chen, Y.; Chen, H.; Ma, M.; Chen, F.; Guo, L.; Zhang, L.; Shi, J. Double Mesoporous Silica Shelled Spherical/ellipsoidal Nanostructures: Synthesis and Hydrophilic/hydrophobic Anticancer Drug Delivery. J. Mater. Chem. 2011, 21, 5290–5298.

(16)

Wu, Z.; Zou, X.; Yang, L.; Lin, S.; Fan, J.; Yang, B.; Sun, X.; Wan, Q.; Chen, Y.; Fu, S. Thermosensitive Hydrogel Used in Dual Drug Delivery System with Paclitaxel-Loaded Micelles for in Situ Treatment of Lung Cancer. Colloids Surf., B. 2014, 122, 90–98.

(17)

Byun, E.; Lee, H. Enhanced Loading Efficiency and Sustained Release of Doxorubicin from Hyaluronic Acid/graphene Oxide Composite Hydrogels by a Mussel-Inspired Catecholamine. J. Nanosci. Nanotechnol. 2014, 14, 7395– 7401.

(18)

Wu, Z.; Zou, X.; Yang, L.; Lin, S.; Fan, J.; Yang, B.; Sun, X.; Wan, Q.; Chen, Y.; Fu, S. Thermosensitive Hydrogel Used in Dual Drug Delivery System with Paclitaxel-Loaded Micelles for in Situ Treatment of Lung Cancer. Colloids Surf., B. 2014, 122, 90–98.

(19)

Zhang, W.; Cui, T.; Liu, L.; Wu, Q.; Sun, L.; Li, L.; Wang, N.; Gong, C. Improving Anti-Tumor Activity of Curcumin by Polymeric Micelles in Thermosensitive Hydrogel System in Colorectal Peritoneal Carcinomatosis Model. J. Biomed. Nanotechnol. 2015, 11, 1173–1182.

(20)

Collett, J. H. Poloxamer. In Handbook of Pharmaceutical Excipients; Rowe, R. C.; Sheskey, P. J.; Quinn, M. E., Eds.; Pharmaceutical Press and APhA:


Page 34 of 37

Page 35 of 37

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


Chicago and London, 2009; pp. 506–509. (21)

Dumortier, G.; Grossiord, J. L.; Agnely, F.; Chaumeil, J. C. A Review of Poloxamer 407 Pharmaceutical and Pharmacological Characteristics. Pharm. Res. 2006, 23, 2709‒2728.

(22)

Harris, L.; Batist, G.; Belt, R.; Rovira, D.; Navari, R.; Azarnia, N.; Welles, L.; Winer, E.; Garrett, T.; Blayney, D.; et al. Liposome-Encapsulated Doxorubicin Compared with Conventional Doxorubicin in a Randomized Multicenter Trial as First-Line Therapy of Metastatic Breast Carcinoma. Cancer 2002, 94, 25–36.

(23)

Paula, A. J.; Montoro, L. A.; Filho, A. G. S.; Alves, O. L. Towards Long-Term Colloidal Stability of Silica-Based Nanocarriers for Hydrophobic Molecules: Beyond the Stöber Method. Chem. Commun. 2012, 48, 591–593.

(24)

Stöber, W.; Fink, A.; Bohn, E. Controlled Growth of Monodisperse Silica Spheres in the Micron Size Range. J. Colloid Interface Sci. 1968, 26, 62–69.

(25)

Cauda, V.; Schlossbauer, A.; Kecht, J.; Zurner, A.; Bein, T. Multiple CoreShell Functionalized Colloidal Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2009, 131, 11361–11370.

(26)

Schmolka, I. R. Artificial Skin I . Preparation and Properties Treatment of Burns. J. Biomed. Mater. Res. 1972, 6, 571–582.

(27)

Brinker, C. J.; Frye, G. C.; Hurd, A. J.; Ashley, C. S. Fundamentals of Sol-Gel Dip Coating. Thin Solid Films 1991, 201, 97–108.

(28)

Anderson, B. C.; Pandit, N. K.; Mallapragada, S. K. Understanding Drug Release from Poly(ethylene Oxide)-B-Poly(propylene Oxide)-B-Poly(ethylene Oxide) Gels. J. Control. Release 2001, 70, 157–167.

(29)

Asakura, S.; Oosawa, F. Interaction between Particles Suspended in Solutions of Macromolecules. J. Polym. Sci. 1958, 33, 183–192.

(30)

McQuarrie, D. Statistical Mechanics; Harper & Row: New York, 1976; p 641.

(31)

Crocker, J.; Matteo, J.; Dinsmore, A.; Yodh, A. Entropic Attraction and Repulsion in Binary Colloids Probed with a Line Optical Tweezer. Phys. Rev. Lett. 1999, 82, 4352–4355.

(32)

Alexandridis, P.; Hatton, T. A. A. Poly(ethylene Oxide)-Poly(propylene Oxide)-Poly (Ethylene Oxide) Block Copolymer Surfactants in Aqueous Solutions and at Interfaces: Thermodynamics, Structure, Dynamics, and Modeling. Colloids Surf., A. 1995, 96, 1–46.

(33)

Batist, B. G.; Ramakrishnan, G.; Rao, C. S.; Chandrasekharan, A.; Gutheil, J.; Guthrie, T.; Shah, P.; Khojasteh, A.; Nair, M. K.; Hoelzer, K.; et al. Reduced Cardiotoxicity and Preserved Antitumor Efficacy of Liposome-Encapsulated Doxorubicin and Multicenter Trial of Metastatic Breast Cancer. 2001, 19, 1444–1454.

(34)

Gabizon, A.; Shmeeda, H.; Barenholz, Y. Pharmacokinetics of Pegylated Liposomal Doxorubicin: Review of Animal and Human Studies. Clin.Pharmacokinet. 2003, 42, 419–436.

(35)

Kim, J.; Yoon, I.; Cho, H.; Kim, D.; Choi, Y.; Kim, D. Emulsion-Based Colloidal Nanosystems for Oral Delivery of Doxorubicin: Improved Intestinal Paracellular Absorption and Alleviated Cardiotoxicity. Int. J. Pharm. 2014, 464, 117–126. 35 Environment ACS Paragon Plus


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

(36)

Swarnakar, N. K.; Thanki, K.; Jain, S. Bicontinuous Cubic Liquid Crystalline Nanoparticles for Oral Delivery of Doxorubicin: Implications on Bioavailability, Therapeutic Efficacy, and Cardiotoxicity. Pharm. Res. 2014, 31, 1219–1238.

(37)

Gabizon, A. A. Stealth Liposomes and Tumor Targeting: One Step Further in the Quest for the Magic Bullet. Clin. Cancer Res. 2001, 7, 223–225.

(38)

Tahover, E.; Patil, Y. P.; Gabizon, A. A. Emerging Delivery Systems to Reduce Doxorubicin Cardiotoxicity and Improve Therapeutic Index: Focus on Liposomes. Anticancer. Drugs 2015, 26, 241–258.

(39)

Mamaeva, V.; Sahlgren, C.; Lindén, M. Mesoporous Silica Nanoparticles in Medicine-Recent Advances. Adv. Drug Deliv. Rev. 2013, 65, 689–702.

(40)

Liu, J.; Luo, Z.; Zhang, J.; Luo, T.; Zhou, J.; Zhao, X.; Cai, K. Hollow Mesoporous Silica Nanoparticles Facilitated Drug Delivery via Cascade pH Stimuli in Tumor Microenvironment for Tumor Therapy. Biomaterials 2016, 83, 51–65.

(41)

Tang, L.; Gabrielson, N. P.; Uckun, F. M.; Fan, T. M.; Cheng, J. SizeDependent Tumor Penetration and in Vivo Efficacy of Monodisperse DrugSilica Nanoconjugates. Mol. Pharm. 2013, 10, 883–892.

(42)

Silveira, C. P.; Apolinário, L. M.; Fávaro, W. J.; Paula, A. J.; Durán, N. Hybrid Biomaterial Based on Porous Silica Nanoparticles and Pluronic F-127 for Sustained Release of Sildenafil: In Vivo Study on Prostate Cancer. RSC Adv. 2015, 5, 81348–81355.

(43)

Guo, M.; Xu, X.; Yan, X.; Wang, S.; Gao, S.; Zhu, S. In Vivo Biodistribution and Synergistic Toxicity of Silica Nanoparticles and Cadmium Chloride in Mice. J. Hazard. Mater. 2013, 260, 780–788.

(44)

Liu, H.; Jia, G.; Chen, S.; Ma, H.; Zhao, Y.; Wang, J.; Zhang, C.; Wang, S.; Zhang, J. In Vivo Biodistribution and Toxicity of Gd2O3:Eu3+ Nanotubes in Mice after Intraperitoneal Injection. RSC Adv. 2015, 5, 73601–73611.

(45)

Kurantowicz, N.; Strojny, B.; Sawosz, E.; Jaworski, S.; Kutwin, M.; Grodzik, M.; Wierzbicki, M.; Lipińska, L.; Mitura, K.; Chwalibog, A. Biodistribution of a High Dose of Diamond, Graphite, and Graphene Oxide Nanoparticles After Multiple Intraperitoneal Injections in Rats. Nanoscale Res. Lett. 2015, 10, 398.

(46)

Yang, K.; Gong, H.; Shi, X.; Wan, J.; Zhang, Y.; Liu, Z. Biomaterials In Vivo Biodistribution and Toxicology of Functionalized Nano-Graphene Oxide in Mice after Oral and Intraperitoneal Administration. Biomaterials 2013, 34, 2787–2795.

(47)

Tarn, D.; Ashley, C. E.; Xue, M.; Carnes, E. C.; Zink, J. I.; Brinker, C. J. Mesoporous Silica Nanoparticle Nanocarriers: Biofunctionality and Biocompatibility. Acc. Chem. Res. 2013, 46, 792–801.

(48)

Mahon, E.; Hristov, D. R.; Dawson, K. A. Stabilising Fluorescent Silica Nanoparticles against Dissolution Effects for Biological Studies. Chem. Commun. 2012, 48, 7970–4972.


Page 36 of 37

Page 37 of 37

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


Table of Contents 47x27mm (300 x 300 DPI)

ACS Paragon Plus Environment

Doxorubicin-Functionalized Silica Nanoparticles Incorporated into a

Recommend Documents