Biomaterials xxx (2014) 1e12

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In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart € lli a, 1, Mo  nica P.A. Ferreira b, 1, Sini M. Kinnunen c, Jaana Rysa € a, d, Marja A. To n Szabo  a, Raisa E. Serpi a, f, Pauli J. Ohukainen a, €kila € b, e, Zolta Ermei M. Ma a €lima €ki , Alexandra M.R. Correia b, Jarno J. Salonen e, Jouni T. Hirvonen b, Mika J. Va lder A. Santos b, * Heikki J. Ruskoaho a, c, **, He a

Department of Pharmacology and Toxicology, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland c Division of Pharmacology and Pharmacotherapy, University of Helsinki, FI-00014 Helsinki, Finland d School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, FI-70211 Kuopio, Finland e Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland f Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 April 2014 Accepted 28 May 2014 Available online xxx

Myocardial infarction (MI), commonly known as a heart attack, is the irreversible necrosis of heart muscle secondary to prolonged ischemia, which is an increasing problem in terms of morbidity, mortality and healthcare costs worldwide. Along with the idea to develop nanocarriers that efficiently deliver therapeutic agents to target the heart, in this study, we aimed to test the in vivo biocompatibility of different sizes of thermally hydrocarbonized porous silicon (THCPSi) microparticles and thermally oxidized porous silicon (TOPSi) micro and nanoparticles in the heart tissue. Despite the absence or low cytotoxicity, both particle types showed good in vivo biocompatibility, with no influence on hematological parameters and no considerable changes in cardiac function before and after MI. The local injection of THCPSi microparticles into the myocardium led to significant higher activation of inflammatory cytokine and fibrosis promoting genes compared to TOPSi micro and nanoparticles; however, both particles showed no significant effect on myocardial fibrosis at one week post-injection. Our results suggest that THCPSi and TOPSi micro and nanoparticles could be applied for cardiac delivery of therapeutic agents in the future, and the PSi biomaterials might serve as a promising platform for the specific treatment of heart diseases. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Biocompatibility Porous silicon Microparticles Nanoparticles Cardiac delivery Myocardial infarction

1. Introduction Nanotechnology has impacted tremendously the medical research with increasing importance in the development of new therapeutic approaches and applications [1]. Among these applications, the use of nanocarriers has gained particular interest in order to achieve successful delivery of therapeutics and imaging

* Corresponding author. Tel.: þ358 2941 59661; fax: þ358 2941 59138. ** Corresponding author. Division of Pharmacology and Pharmacotherapy, University of Helsinki, Finland. E-mail addresses: heikki.ruskoaho@helsinki.fi (H.J. Ruskoaho), helder.santos@helsinki.fi (H.A. Santos). 1 These authors contributed equally to this work.

agents for the treatment and diagnostics of different diseases, such as cancer [2e7] and cardiovascular diseases [8,9]. Chronic heart failure (HF) is a complex clinical syndrome derived from multiple causes that arise from secondary to inherited or acquired abnormalities of cardiac structure and/or function [10,11]. Myocardial infarction (MI) continues to be an increasing problem in terms of morbidity, mortality and healthcare costs [12]. After the event of MI, a series of complex remodeling responses lead to alterations in the cardiomyocyte structure and/or function through coordinated changes in gene and protein expression [13]. Hypertrophic growth is the primary mechanism by which the heart reduces stress on the ventricular wall by enlarging individual myocytes to augment cardiac pump function and decrease ventricular wall tension [14e16]. Currently, well-known treatments for

http://dx.doi.org/10.1016/j.biomaterials.2014.05.078 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078

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€lli et al. / Biomaterials xxx (2014) 1e12 M.A. To

HF using pharmacologic agents have proven to be effective in reducing hypertrophy and/or negative remodeling of the myocardium, such as angiotensin-converting-enzyme inhibitors, angiotensin-receptor antagonists, mineralocorticoid receptor antagonists, calcium-channel blockers and beta-blockers [11,15]. Additionally, biventricular pacing and coronary bypass surgery have been shown to reduce the rate of hospitalizations significantly, and thus, minimizing the mortality or improving the functional status of the heart [11]. Despite optimal treatment with existing drugs, the overall efficacy of these therapeutic approaches is rather limited in many cases, and also the possible deleterious effects of these therapeutic agents must be considered when used at doses required for the desired therapeutic effect [11,15,17]. Therefore, novel therapeutic strategies are urgently necessary to prevent such high mortality rates resulting from MI. One approach for the abovementioned problems can be the efficient delivery of therapeutic agents to the unhealthy heart tissue. Different nanocarriers have recently been shown to efficiently deliver drugs into the injured myocardium [8,9,16,18]. Recently, gene delivery approaches have also been employed for the treatment of HF, showing promising results concerning the improvement of cardiac function, as well as prevention of the left ventricular diastolic dysfunction [17,19]. Thus, we hypothesize here that porous silicon (PSi) can also act as a biomaterial for targeted drug delivery to the heart. PSi has been widely studied for numerous applications, particularly for the efficient targeting to specific tissues using functionalized PSi nanoparticles [20e23], and its potential in the biomedical field has already been demonstrated. For example, the cytocompatibility of PSi has been demonstrated with modified PSi nanoparticles with intestinal (HT-29), liver (HepG2), macrophage (RAW 264.7), and stomach (AGS) cell lines [24,25]. The physicochemical properties of PSi such as particle size, shape, pore size, large surface area (>300 m2/ g), high degree of porosity (50e80%) and chemical surface [2,26,27], render the material special characteristics to improve the solubility of poorly water soluble drugs [28e30], and thus, ideally for controlled drug release [24,31,32]. Additionally, PSi has also been demonstrated to be biodegradable [33e36], and in vivo studies have shown its biocompatibility also in living tissues [37e40]. The biosafety of different biomaterials is therefore of utmost importance in order to guarantee the successful drug delivery to the target site with minimal or none side effects [41]. In this respect, nano/microtoxicology studies are extensively conducted in order to evaluate the biosafety and possible side effects of the carriers on healthy and injured organs or tissues [41e44]. Despite the vast research on the materials' toxicology, little is still known about the effects of nanostructured materials on the heart tissue [45], hence more efforts should be put in this area of research. Herein, based on our previous studies with PSi particles, and as a result of the particles' stability and their physicochemical properties, as well as due to the lack of previous reported studies with PSi and cardiomyocyte cells and heart tissues, we aimed to test both the in vitro cytocompatibility in primary cardiomyocyte cells and the in vivo biocompatibility of different sizes of hydrophobic PSi microparticles and hydrophilic PSi micro and nanoparticles in the heart tissue. PSi micro and nanoparticles were injected directly into the normal rat myocardium and also in the infarcted rat heart, and the function of the heart post-injection was assessed by echocardiography. The effect of the intramyocardial PSi particle injections on rat hematological values was analyzed. The alterations in the expression of inflammatory related and pro-fibrotic genes in the injection area of the hearts were also evaluated. Inflammatory and fibrotic responses at the tissue level were measured by immunohistology.

2. Materials and methods 2.1. Preparation of thermally hydrocarbonized porous silicon (THCPSi) and thermally oxidized porous silicon (TOPSi) micro and nanoparticles Based on our previous studies with PSi particles and as a result of the particles' stability in solution and their physicochemical properties, we have chosen two different sizes of THCPSi and TOPSi microparticles, and one type of TOPSi nanoparticles. THCPSi and TOPSi were prepared electrochemically as described elsewhere [29,46,47]. Briefly, free-standing PSi films were electrochemically anodized from monocrystalline, boron doped pþ Si 〈100〉 wafers with a resistivity of 0.01e0.02 U .cm using a 1:1 (v/v) hydrofluoric acid (38%)‒ethanol (EtOH) electrolyte. For the microparticles, the free-standing films were produced using a constant etching current. With the nanoparticles, multilayer PSi films were formed by applying a repeating low/high pulse etching current profile. The porous films were lifted off from the Si substrates by increasing the etching current abruptly to the electropolishing region. The TOPSi nanoparticles were produced by thermally oxidizing the multilayer films in ambient air for 2 h at 300  C, followed by a wet milling in EtOH, with the final size selection done using centrifugation. To produce microparticles, the anodized films were dry milled and then the particles were separated into different size classes by sieving followed by centrifugation. In the case of THCPSi, the milled microparticles were initially immersed into a hydrofluoric acid solution to remove the native oxide formed during the milling and dried. The obtained hydrogen terminated PSi microparticles were then thermally hydrocarbonized under a 1:1 (vol.) C2H2eN2 flow for 15 min at room temperature (RT) followed by a heat treatment for 15 min at 500  C and cooled back to RT under N2 flow [47]. The size separation was obtained by the same method of sieving and centrifugation described for TOPSi microparticles. 2.2. Physicochemical characterization of THCPSi and TOPSi micro and nanoparticles The surface chemistry of the THCPSi and TOPSi particles was characterized with Fourier transform infrared spectroscopy (FTIR) using a Mattson Galaxy 6020 (Mattson Instruments, USA) equipped with a HgCdTe detector cooled to 196  C and a diffuse reflectance accessory (Spectra-Tech Inc., USA). The porous properties of the different PSi samples were characterized by N2 sorption at 196  C with a TriStar 3000 (Micromeritics Inc., USA). The specific surface area was calculated using the BrunauereEmmetteTeller (BET) theory, and the total pore volume was determined from the isotherm using the adsorption value at relative pressure of p/p0 ¼ 0.97. The average pore diameter was calculated by assuming the pores as cylindrical and using the obtained BET area and total pore volume. The average size of THCPSi and TOPSi microparticles was measured using scanning electron microscope (SEM; Zeiss DSM 962). The samples were suspended in EtOH and a droplet of each sample was added to a metallic sample holder, allowed to dry, and sputter coated with platinum before imaging. The average size of the TOPSi nanoparticles was measured from transmission electron microscope (TEM; FEI Tecnai F12) images. The samples for TEM were prepared by adding droplets of the TOPSi nanoparticles suspensions (prepared in EtOH) on copperecarbon grids and allowed to dry overnight. 2.3. Preparation of the PSi micro and nanoparticles for cytotoxicity tests and primary cardiomyocyte culture For the viability tests, 10 mM Hank's balanced salt solution (HBSS)4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution at pH 7.4 was freshly made and the suspensions of micro and nanoparticles were prepared at concentrations ranging from 25 to 1000 mg/mL. The cell culture reagents were obtained from SigmaeAldrich, unless otherwise stated. Primary cultures of neonatal rat ventricular cardiomyocytes were prepared from 2 to 4-day-old Wistar rats [48]. The animals were sacrificed by decapitation, and the ventricles were excised and cut into small pieces. The ventricle pieces were incubated at 37  C by shaking during 2 h in a solution containing 100 mM NaCl, 10 mM KCl, 1.2 mM KH2PO4, 4.0 mM MgSO4, 50 mM taurine, 20 mM glucose, 10 mM HEPES, 2 mg/mL collagenase type 2 (Worthington, Lakewood), 2 mg/mL pancreatin and 1% penicillinestreptomycin (PS, Gibco). After incubation the detached cells were collected into a 15 mL tubes and centrifuged for 5 min at 160 g. The supernatant and the top layer of the pellet containing damaged cells were discarded and the isolated cardiomyocytes were

Table 1 Physical characterization of the PSi particles: average pore diameter (D), total pore volume (V; p/p0 ¼ 0.97), and specific surface area (As). Sample size THCPSi 7 and 19 mm TOPSi 7 and 17 mm 110 nm

D (nm)

V (cm3/g)

As (m2/g)

13.7 ± 0.2

1.16 ± 0.01

337 ± 8

12.7 ± 0.1 13.3 ± 0.6

0.61 ± 0.01 0.59 ± 0.03

193 ± 2 176 ± 4

€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078

€lli et al. / Biomaterials xxx (2014) 1e12 M.A. To

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Fig. 1. SEM images of TOPSi 7 mm (A), TOPSi 17 mm (B), THCPSi 7 mm (C), THCPSi 19 mm (D) and TEM image of TOPSi 110 nm nanoparticles (E) used in this study, and their respective size distribution. Scale bars are 40 mm and 500 nm for the SEM and TEM images, respectively.

€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078

€lli et al. / Biomaterials xxx (2014) 1e12 M.A. To

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and treated similarly as described above. The luminescence was measured using a Varioskan Flash Multimode Reader (Thermo Fisher Scientific). All the experiments were carried out at least in triplicate. 2.5. Particle injections into the hearts

Fig. 2. The FTIR spectra of the THCPSi (i) and the TOPSi (ii) particles. The spectra have been normalized and shifted vertically for clarity.

About 8-week old male SpragueeDawley rats weighing 250e300 g were anesthetized with medetomidine hydrochloride (Domitor, Orion Pharma, 250 mg/kg administered intraperitoneally, i.p.) and ketamine hydrochloride (Ketaminol, Intervet International B.V., 50 mg/kg, i.p.). A left thoracotomy and pericardial incision was performed to expose the heart and single injections of a vehicle (100 mL phosphate buffer solution, PBS (Sigma), for TOPSi and THCPSi microparticles; 100 mL PBS/EtOH 3.6% for TOPSi nanoparticles) or micro/nanoparticles (TOPSi microparticles 500 mg in 100 mL PBS; THCPSi microparticles 500 mg in 100 mL PBS/10% EtOH; and TOPSi nanoparticles 250 mg in 100 mL PBS/EtOH 3.6%) were injected directly into the anterior wall of the left ventricle (LV) using a Hamilton precision syringe. The syringe was inserted into one site of the LV free wall (apex to base), and then slowly the solution was injected while withdrawing the syringe. The heart was repositioned, the rat was briefly hyperventilated and the incision closed. After the operation, anesthesia was partially antagonized with atipamezole hydrochloride (Antisedan, Orion Pharma, 1.5 mg/kg, i.p.). For post-operative analgesia, buprenorphine hydrochloride (Vetergesic, Orion Pharma, 0.05e0.2 mg/kg administered subcutaneously, s.c.) twice daily and carprofen (Rimadyl, Pfizer, 5 mg/kg, s.c.) once daily for three days were used. At one week or 4 weeks post-injection the echocardiography was performed, the rats were decapitated and particular tissues were collected for further analysis. 2.6. Experimental model of myocardial infarction (MI)

suspended in Dulbecco's Modified Eagle Medium (DMEM)/F12 culture medium (Gibco) containing 2.5 mM L-glutamine, supplemented with 1% PS and 10% fetal bovine serum (Gibco). Cells were pre-plated for 30e60 min on 10 cm diameter cell culture plates, and unattached cardiomyocytes were collected with the medium. The amount of viable cells was counted with the help of a light-microscope in a Burger's chamber. Cardiomyocytes were seeded into 96-well plates (Corning) at a density of 5  105 cells/well, according to the assay used. After 24 h incubation, the medium was replaced with complete serum free medium (DMEM/F12, 2.5 mg/mL bovine serum albumin, 1 mM insulin, 2.5 mM L-glutamine, 32 nM selenium, 2.8 mM sodium pyruvate, 5.64 mg/mL transferrin, 1 nM T3, and 100 IU/mL PS). Cells were cultured another 24 h before performing the tests. The temperature in the culture chamber was 37  C with 5% CO2 and 95% air atmosphere.

2.4. Cell viability assay In this assay, the metabolically active cells were quantified based on the amount of ATP produced by viable cells (CellTiter-Glo® Luminescence Cell Viability Assay, Promega). Therefore, the amount of ATP was directly proportional to the number of living cells present in the culture. Once the primary cardiomyocytes were attached to the 96-well plates, the wells were washed twice with 100 mL of 10 mM HBSSeHEPES (pH 7.4). After washing, 100 mL of the PSi suspensions (in HBSS) at concentration of 25e1000 mg/mL were added to each well. The cells were then incubated at 37  C for 3 h. After incubation, the wells were washed twice with HBSS to remove the excess of particles and the plates were equilibrated at RT for 30 min. After that, the assay reagent was added to each well. A positive (1% Triton X-100 solution) and negative (10 mM HBSSeHEPES solution) control wells were also used

Acute MI was produced by ligation of the LAD (left anterior descending) during medetomidine hydrochloride and ketamine hydrochloride anesthesia as previously described [49]. The rat was connected to the respirator through a tracheotomy and ventilated at a rate of 55e60 breaths/min. A left thoracotomy and pericardial incision were performed and the LAD was ligated about 3 mM from its origin. After ligation of the LAD, the heart was repositioned in the chest and the incision was closed. The anesthetic effects were antagonized with atipamezole hydrochloride and the rats were hydrated with 5 mL physiological saline solution. Buprenorphine hydrochloride 0.1 mg/kg twice daily and carprofen 5 mg/kg once daily were administered s.c. for 3 day as a post-operative analgesia. The sham-operated rats underwent the same surgical procedure without the ligation of LAD [50]. TOPSi 7 mm or 110 nm particles were injected into the anterior wall of the LV before the ligation of LAD. The TOPSi particles delivery to the sham-operated hearts was performed using the same technique without the ligation of LAD. At one week post-infarction the echocardiography was performed, the rats were decapitated and particular tissues were collected for further analysis. 2.7. Echocardiographic measurements Transthoracic echocardiography was performed using the Acuson Ultrasound System (Sequoia™ 512) and a 15-MHz linear transducer (15L8) (Acuson, Mountain View, CA, USA). Before examination, rats were sedated with Ketamine (50 mg/kg administered i.p.) and xylazine (10 mg/kg administered i.p.). Using two-dimensional imaging, a short axis view of the LV at the level of the papillary muscles was obtained, and a two dimensionally guided M-mode recording through the anterior and posterior walls of the LV was obtained. LV end-systolic and end-diastolic dimensions

Fig. 3. ATP production of primary cardiomyocyte cells determined by the CellTiter-Glo® Luminescence Cell Viability Assay after 3 h incubation at 37  C with THCPSi and TOPSi microparticles (A), and TOPSi nanoparticles (B). The results are expressed as mean ± SEM (n ¼ 3). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control.

€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078

€lli et al. / Biomaterials xxx (2014) 1e12 M.A. To as well as the thickness of the interventricular septum and posterior wall were measured from the M-mode tracings. LV fractional shortening (FS) and ejection fraction (EF) were calculated from the M-mode LV dimensions using equations (1) and (2): Fractional shortening ð%Þ ¼

Ejection fractionð%Þ ¼

ðLVEDD  LVESD Þ  100 LVEDD

ðLVEDD Þ3  ðLVESD Þ3 ðLVEDD Þ3

(1)

 100

(2)

where LVEDD is the left ventricular end-diastolic diameter and LVESD the left ventricular end-systolic diameter. An average of three measurements of each variable was used. All echocardiographic measurements were performed by skilled sonographer blinded to the treatments. After echocardiography, the animals were euthanized by decapitation, blood samples were collected and the hearts were removed and weighed. The injection area, middle part of the heart, was used for histological analysis and the apex was immersed in liquid nitrogen and stored at 70  C for further analysis. 2.8. Hematological analysis Terminal blood samples were drawn during the decapitation of the animals. Blood was collected in sodium ethylenediaminetetra-acetic acid tubes, and the red and white blood cells (RBC, WBC) were counted. The determination of the hemoglobin level (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW), platelets (PLT), mean platelet volume (MPV), mean corpuscular hemoglobin concentration (MCHC), mean cellular volume (MCV) and white blood cell distribution (NEU, neutrophils; LYM, lymphocytes; MO, monocytes; EO, eosinophils; BA, basophils) were performed with Cell-Dyn Sapphire (Abbott). 2.9. Immunohistochemistry After sacrificing the animals, the hearts were removed and transverse sections of the middle part of the heart were fixed in 10% neutral buffered formalin for 1e2 days, embedded in paraffin, cut into 5 mm-sections from the injection area and mounted on slides. To evaluate the presence of micro/nanoparticles and inflammatory response, formalin-fixed, paraffin-embedded sections were deparaffinized in xylene and dehydrated in graded EtOH and stained with hematoxylin and eosin (H&E). Inflammation was scored from the H&E stained slides by evaluating the size of the inflamed granulation tissue area as either negative () or positive (þ); no inflammation or very slight inflammation was scored as negative, substantial inflammation was scored as positive. Masson's trichrome technique was used to define the extent of fibrosis (fibrotic area/total LV area) in the LV by using Nikon NIS-

5

Elements 3.2 software. Immunohistological analysis was performed by using a light microscope (Nikon Eclipse 50i). 2.10. Gene expression analysis The total RNA from the apical heart tissue was isolated by the guanidine thiocyanateeCsCl method [51]. For quantitative real-time polymerase chain reaction (RT-PCR) analyses, cDNA was synthesized from total RNA with a First-Strand cDNA Synthesis Kit (GE Healthcare Life Sciences) following the manufacturer's protocol. RNA was analyzed by RT-PCR on an ABI 7300 sequence detection system (Applied Biosystems) using TaqMan chemistry. The results were quantified using the DDCT method and normalized to 18S housekeeping gene from the same samples to correct the potential variation in sample loading. The sequences of the forward and reverse primers and fluorogenic probes for RNA detection are shown in the Supplementary Information Table S1. 2.11. Statistics Results are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using SPSS version 19.0.0.1 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 5.00 (GraphPad Software, San Diego, California, USA). To determine the statistical difference between two groups, the independent samples Student's t-test was used. For multiple comparisons, the statistical significance was evaluated by one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) post hoc test. For evaluation of the extent of inflammation and granulation tissue caused by the micro/nanoparticle injection, Fisher's exact test was used. The probability values of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant. 2.12. Ethics All experimental protocols with animals were approved by the Animal Use and Care Committee of the University of Oulu and the Regional State Administrative Agency for Southern Finland, and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

3. Results and discussion 3.1. Physicochemical characterization of THCPSi and TOPSi micro and nanoparticles The structural characteristics of the THCPSi and TOPSi particles were determined using N2 sorption, and the results shown in

Table 2 Effect of intramyocardial delivery of THCPSi and TOPSi micro and nanoparticles in rat on hematological values at one week post-injection. The results are expressed as mean ± SEM (n ¼ 3e4). *p < 0.05 vs. control. Mean ± SEM WBC (109/L)

HGB (g/L)

PLT (109/L)

NEUT (109/L)

Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm

12.5 12.1 12.5 11.4 10.8 13.4 15.1 150 148 146 148 144 157 153 650 755 599 745 632 710 622 3.9 3.2 3.0 3.6 3.1 3.8 5.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 1.7 3.1 1.9 1.7 1.5 2.0 3 3 4 2 3 1 2 46 63 80 48 86 61 127 0.8 0.9 0.7 1.1 0.7 1.0 1.3

Mean ± SEM LYMPH (109/L)

MONO (109/L)

EO (109/L)

BASO (109/L)

Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm

7.7 8.1 5.9 7.1 7.1 8.7 8.0 0.83 0.75 0.48 0.67 0.48 0.82 1.50 0.11 0.05 0.06 0.02 0.08 0.02 0.03 0.01 0.01 0.01 0.01 0.01 0.00 0.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.5 0.2 0.9 1.3 0.6 1.2 0.16 0.27 0.14 0.16 0.05 0.13 0.40 0.05 0.02 0.02 0.02* 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

BASO, basophils; EO, eosinophils; HGB, hemoglobin; LYMPH, lymphocytes; MONO, monocytes; NEUT, neutrophils; PLT, platelets; WBC, white blood cells; *p < 0.05.

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Table 3 Immunohistological analysis revealing an increased inflammatory response in THCPSi 7 mm microparticle treated rat heart tissues compared to THCPSi 19 mm, TOPSi micro/nanoparticle and control treated heart tissue sections one week postinjection. Inflammation was scored from the H&E stained slides by evaluating the size of the inflamed granulation tissue area as either negative () or positive (þ); no inflammation or very slight inflammation was scored as negative, substantial inflammation was scored as positive. Fibrosis (% fibrotic area of total LV area) in the THCPSi and TOPSi micro/nanoparticle-injected hearts did not differ significantly from the control treatments. *p < 0.05 vs. control. Treatment

Inflammation

Fibrosis %

Control THCPSi 7 mm THCPSi 19 mm TOPSi 7 mm TOPSi 17 mm Control TOPSi 110 nm

e þ* e e e e e

5.95 7.76 5.53 6.55 7.79 9.46 11.38

± ± ± ± ± ± ±

2.55 1.52 1.82 2.74 5.40 3.61 3.86

Presence of particles No particles observed Particles form a band Particles form a band No particles observed No particles observed No particles observed No particles observed

Table 1. Fig. 1 shows the SEM pictures of THCPSi and TOPSi microparticles and the TEM picture of TOPSi nanoparticles. In general, both the micro and nanoparticles showed an irregular shape, with an average size of 7 and 19 mm for the THCPSi microparticles, 7 and 17 mm for the TOPSi microparticles, and 110 nm for the TOPSi nanoparticles. The chemical structure of the surface of the THCPSi and TOPSi particles was evaluated with FTIR (Fig. 2). The FTIR spectra of the THCPSi particles presented several distinctive features, such as the symmetric and asymmetric CH2 and CH3 stretching bands between

2860 and 2970 cm1. The vinyl CeH stretching band at 3055 cm1 is also clearly visible along with a band at 1595 cm1, assigned to SieC]C stretching vibrations. In addition, the broad absorption band centered at 1000 cm1 is characteristic for the THCPSi [52]. The TOPSi particles showed a broad band representative of the SieO stretching vibrations between 1000 and 1200 cm1 confirming the thermal oxidation of the particles. Furthermore, characteristic n(O3SieH) stretching vibrations at 2270 cm1 were observed, indicating the remaining Si hydrides on the surface are mainly backbond oxidized. The shoulder band at 3740 cm1 is indicative of the presence of SieOH species, also confirming the thermal oxidation treatment of TOPSi particles.

3.2. Cell viability In vitro nano/microtoxicology studies are crucial in order to predict the biosafety of the carriers. Based on our previous studies with PSi particles, and as a result of the particles' stability and their physicochemical properties, we evaluated the cytotoxicity in two different sizes of THCPSi and TOPSi microparticles, and one type of TOPSi nanoparticles [26,31,39,55]. The viability of primary cardiomyocytes was calculated taking into account their metabolic activity, by measuring the ATP production in presence of THCPSi and TOPSi microparticles, as well as TOPSi nanoparticles, using a luminescence-based assay (Fig. 3A) [46,53]. After 3 h incubation time with primary cardiomyocytes, the lower concentrations of THCPSi microparticles did not induce significant toxicity, with very

Fig. 4. Effect of intramyocardial delivery of THCPSi or TOPSi micro and nanoparticles on cardiac function in normal rat heart. THCPSi and TOPSi microparticles had no effect on the ejection fraction (A), fractional shortening (B), stroke volume (C) or cardiac output (D) at one week or 4 weeks post-injection. TOPSi 110 nm particles slightly increased the ejection fraction and fractional shortening (A and B) at one week. The results are expressed as mean ± SEM (n ¼ 3e4). *p < 0.05 vs. control.

€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078

€lli et al. / Biomaterials xxx (2014) 1e12 M.A. To

high and concentration-dependent ATP content observed for all particle concentrations. For the highest concentration, there was a statistically significant decrease of ATP content to 85% for THCPSi 7 mm (p < 0.05). Also, statistically significant decrease of ATP content is observed for the two highest concentrations of THCPSi 19 mm (p < 0.01 and p < 0.001). TOPSi microparticles induced a statistically significant decrease in cell ATP content (p < 0.01 and p < 0.001), which appears to be concentration-dependent, being more pronounced at higher concentrations. Particle size-dependent effects were observed for TOPSi 7 and 17 mm at the concentrations studied, where the bigger particles showed less toxicity to the cells than the smaller ones. The same assay was performed with TOPSi nanoparticles (Fig. 3B). In general, TOPSi nanoparticles have shown to be less toxic than TOPSi microparticles in primary cardiomyocytes, which is in agreement with previous studies, even though other cell lines were used. Our previous work demonstrates that TOPSi particles in a size range of 1e25 mm were found to induce the most toxic effect in the Caco-2 and RAW 264.7 macrophage cells, compared to TOPSi nanoparticles [29]. It was found that size and shape can influence the route of internalization of particles by the cell, as well as their interaction with the cell wall. The different size and very irregular shape that TOPSi microparticles display compared to TOPSi nanoparticles, which have a tendency to be more spherical, could also be an explanation for the different degree of toxicity observed [54]. It must be taken into account that primary cardiomyocytes have superior sensitivity to harsh conditions compared to continuous cell lines. In general, of all materials studied, THCPSi microparticles and TOPSi nanoparticles were less toxic to the primary cardiomyocytes.

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response to TOPSi 110 nm particle injections (Fig. 4A and B). Cardiac output, stroke volume, heart rate, LV mass and the heart-weight-tobody-weight ratio of the THCPSi or TOPSi particle-injected animals were similar to those of control treated groups (Figs. 4 and 5). Body

3.3. Effect of the THCPSi and TOPSi micro and nanoparticles on the hematological parameters The in vivo biocompatibility of THCPSi and TOPSi micro and nanoparticles was evaluated by injecting the particles into the myocardium of the rats. Blood samples from the control rats and the nano/microparticle injected rats were collected during the decapitation at one week post-injection, and the hematological parameters were measured. There were no statistically significant changes on the hemoglobin levels (HGB), platelets (PLT) and white blood cell counts (WBC) when comparing the THCPSi or TOPSiinjected animals to the control treated rates (Table 2). Similarly, no difference in WBC distribution (NEU, LYM, MO, EO, BA) was seen between the control animals and particle-injected animals with the exception of eosinophil count, which was significantly decreased (81%, p < 0.05) after intramyocardial injection of TOPSi 7 mm (Table 3). No difference between the THCPSi- or TOPSi-injected rats and the control treated animals were seen in the hematocrit (HCT), mean corpuscular hemoglobin (MCH), red cell distribution width (RWD), mean platelet volume (MPV), mean corpuscular hemoglobin concentration (MCHC) and mean cellular volume (MCV) values (Supplementary Information Table S2). 3.4. Cardiac function after THCPSi and TOPSi injections into the normal myocardium To study the effects of THCPSi and TOPSi micro and nanoparticles on the function of the heart after injection into the myocardium, cardiac function was assessed using echocardiography at 7 days or 4 weeks post-injection, before decapitating the animals. THCPSi 7 and 19 mm and TOPSi 7 and 17 mm microparticle injections had no effect on LV systolic function (ejection fraction or fractional shortening) at one week or at 4 weeks compared to control injected animals (Fig. 4A and B). LV ejection fraction (11%) and fractional shortening (13%) increased slightly at one week in

Fig. 5. Heart rate (A), LV mass (B) or heart-to-body-weight ratio (C) at one week or 4 weeks post-injection were not altered in response to intramyocardial delivery of THCPSi or TOPSi micro and nanoparticles into the normal rat heart. The results are expressed as mean ± SEM (n ¼ 3e4).

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weight changes over the follow-up period were not observed (Supplementary Information Fig. S1). Overall, neither THCPSi nor TOPSi microparticles altered the cardiac function after direct injections into the myocardium of normal rat hearts, whereas TOPSi nanoparticles slightly increased the LV systolic function.

weight ratio increased slightly, when comparing infarcted hearts to the sham-operated hearts in both the TOPSi 7 mm and 110 nm particle-injected rats (Fig. 6D). Thus, myocardial treatment with the TOPSi particles did not attenuate functional alterations after MI in rats, suggesting that this type of biomaterial can be used in experimental MI models.

3.5. Cardiac function after TOPSi injections into the myocardium of infarcted rat hearts

3.6. Immunohistological analysis

In order to exclude both the beneficial or injurious effect of TOPSi particles to the cardiac function after acute MI, as well as to evaluate whether the TOPSi particles can be a useful biomaterial in an experimental MI, LAD ligation was performed immediately after intramyocardial particle injections. Ligation of LAD caused a statistically significant decrease in the ejection fraction (Fig. 6A) and fractional shortening (Fig. 6B) in conjunction with the injection of TOPSi 7 mm during the one week's follow-up. The same trend was observed with the TOPSi 110 nm particle injections, although the changes were not statistically significant (Fig. 6A and B). The heart rate was not altered in consequence to any of the aforementioned treatments (Fig. 6C). As expected, the heart-weight-to-body-

Histological sections were made from the THCPSi and TOPSi micro and nanoparticle-injected rat's hearts and stained with H&E for inflammation and with Masson's trichrome for fibrosis. At one week time point, fibrosis in the THCPSi and TOPSi micro/ nanoparticle-injected hearts did not differ significantly from the control hearts (Table 3 and Fig. 7A). On the other hand, increased inflammation in the myocardium after injection of THCPSi 7 mm microparticles were observed, while only a minor inflammatory reaction was seen in control or THCPSi 19 mm and TOPSi treated heart tissue sections (Table 3 and Fig. 7B). The particles injected into the myocardium of the rat hearts were observed in the tissue sections one week post-injections. In the heart tissue sections of

Fig. 6. Effect of intramyocardial delivery of TOPSi micro and nanoparticles on cardiac function in the rat heart after MI at one week post-infarction and particle treatment. (A) Ejection fraction and (B) fractional shortening alterations were normal for an infarcted heart. TOPSi particles had no effect on heart rate (C) or heart-to-body-weight ratio (D) after MI. The results are expressed as mean ± SEM (n ¼ 5e6). *p < 0.05 vs. SHAM.

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Fig. 7. A. Intramyocardial delivery of THCPSi or TOPSi micro and nanoparticles had no significant effect on myocardial fibrosis at one week post-injection. Fibrotic area was assessed from Masson's trichrome-stained LV sections from the rat heart, and is expressed as mean ± SEM (n ¼ 3e4). B. Local THCPSi 7 mm but not THCPSi 19 mm, TOPSi 110 nm, TOPSi 7 mm and TOPSi 17 mm or control treatment increased myocardial inflamed granulation tissue (arrow) in the normal rat heart when injected into the LV myocardium. Paraffin-embedded histological sections were stained with H&E to define the area of inflammation at one week post-injection (scale bar 100 mm).

the injected THCPSi 7 and 19 mm, the particles constituted distinct zones, while only few separate undissolved particles could be observed in TOPSi 7 mm, 17 mm and 110 nm injected sections one week post-injections (Fig. 7B). That demonstrates the better stability of THCPSi particles in the myocardium.

3.7. Activation of inflammatory gene expression in response to THCPSi and TOPSi micro- and nano-particle injections Interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) are proinflammatory cytokines secreted by macrophages in the

Fig. 8. Local THCPSi delivery increased the myocardial expression of inflammatory and pro-fibrotic genes in normal rat heart. TOPSi particles had less effect on cardiac gene expression. (A) IL-6 mRNA levels, (B) TNF-a mRNA levels, (C) Collagen (Col) Ia1 mRNA levels, and (D) Osteopontin (OSP) mRNA levels at one week or 4 weeks post-injection. The results are expressed as mean ± SEM (n ¼ 3e4). *p < 0.05 vs. control.

€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078

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myocardium as a response to tissue injury. IL-6 and TNF-a play important role in stimulating immune response and mediating inflammation as well in modulating tissue repair and adaptation after injury [55]. To evaluate the inflammatory response on a gene level, the mRNA levels of IL-6 and TNF-a genes in the THCPSi and TOPSi micro and nanoparticle-injected rat hearts were determined from the apical tissue samples one week or 4 weeks after particle injections. THCPSi 19 mm treatment increased the expression of both IL-6 and TNF-a gene at one week after injections, while the increase was almost or totally normalized at 4 weeks (Fig. 8A and B). THCPSi 7 mm only slightly increased the IL-6 expression at one week, but the expression of TNF-a gene increased to 4.7-fold compared to control (Fig. 8A and B). TOPSi 7 and 17 mm, and 110 nm particles had no effect on IL-6 and TNF-a gene expression levels at one week post-injection (Fig. 8A and B). Consistently with the immunohistological findings (Table 3), these results showed more pronounced inflammatory response in THCPSi 7 mm microparticle treated rat hearts compared to TOPSi particle treatments or the control treatments. 3.8. Activation of the fibrosis promoting genes in response to THCPSi and TOPSi micro and nanoparticle injections A collagen network of the myocardium, composed largely of types I and III collagens, has multiple functions including a

preservation of tissue architecture and chamber geometry. Fibrosis is a consequence of accumulation of fibrillar collagen into the myocardium [56]. Cytokines play an important role in regulating collagen deposition. Osteopontin is a profibrogenic extracellular matrix protein and cytokine, which promotes collagen synthesis and accumulation [55]. The activation of fibrosis promoting genes collagen Ia1 and osteopontin in THCPSi and TOPSi particle injected rat hearts were analyzed one week and 4 weeks after injections. THCPSi 7 and 19 mm particle injections to the rat myocardium increased collagen Ia1 gene levels to 2.4- and 2.6fold, respectively, compared to control injected rat hearts at one week post-injections, but not at 4 weeks post-injection (Fig. 8C). TOPSi 7 and 17 mm, and 110 nm particles did not alter the collagen Ia1 gene expression (Fig. 8C). THCPSi 7 and 17 mm microparticles increased the osteopontin gene levels to 79- and 14-fold, respectively, compared to the control injected rat hearts at one week post-injections, and at 4 week the osteopontin gene level was normalized (Fig. 8D). TOPSi 7 and 17 mm, and 110 nm particles increased the osteopontin mRNA to 8.1-, 3.2-, and 6.9-fold, respectively, compared to the control treatments at one week post-injection (Fig. 8D). Although the histological analysis did not reveal significant difference in fibrosis between the PSi particle treated hearts and the control treated hearts, the gene expression alterations demonstrated that THCPSi microparticles induced slightly greater expression of pro-fibrotic genes than the control

Fig. 9. The expression of inflammation and pro-fibrotic genes in the rat heart after MI. TOPSi particles had no effect on gene expression alterations after MI. (A) IL-6 mRNA levels, (B) TNF-a mRNA levels, (C) Collagen (Col) Ia1 mRNA levels, and (D) Osteopontin (OSP) mRNA levels at one week post-infarction and particle treatment. The results are expressed as mean ± SEM (n ¼ 5e6). *p < 0.05 and **p < 0.01 vs. SHAM.

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treatment or TOPSi micro/nanoparticles in the rat's heart at one week after treatment, normalizing then at 4 weeks. 3.9. Gene expression changes in infarcted rat hearts in response to TOPSi micro and nanoparticle injections

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.078. References

The inflammatory response and cytokine production play a significant role after MI. Cytokines, such as TNF-a and IL-6, mediate cardiac repair and remodeling through activating matrix metalloproteinase and type I and III collagen formation, integrin regulation, angiogenesis and progenitor cell mobilization [55e57]. Atrial natriuretic peptide (ANP) is a cardiac hormone, which secretion is markedly up-regulated during heart failure [58]. Thus, to assess the effect of TOPSi 7 mm and 110 nm particles on the gene expression after MI, rats were subjected to MI and then the PSi particles were injected into the myocardium. At one week after MI, changes in the gene expression were normal for the infarcted heart. A 4.3-fold ANP mRNA increase was observed in infarcted hearts compared to sham-operated hearts (p < 0.001, Supplementary Information Fig. S2) after injecting TOPSi 7 mm particles into the myocardium of the rats. Furthermore, a significant increase in IL-6 (Fig. 9A), collagen Ia1 (Fig. 9C) and osteopontin (Fig. 9D) mRNA was observed in the TOPSi 7 mm injected rat hearts one week after MI. IL-6, Col Ia1 and OSP gene expressions were increased also in the TOPSi 110 nm-injected rat hearts at one week after MI, although these changes were not statistically significant (Fig. 9). TNF-a mRNA levels did not differ significantly between treatments (Fig. 9B). These gene expression data is in agreement with the previously presented functional data achieved from infarcted rat hearts, indicating that TOPSi micro and nanoparticles can be considered as a useful biomaterial for the treatment of MI in rats. 4. Conclusions PSi micro and nanoparticles were investigated to be used as a potential biomaterial for drug delivery into the heart tissue and for the treatment of MI. Although both particle types were noncytotoxic and showed good in vivo biocompatibility, they differ in the in vivo inflammation and fibrosis promoting responses. Local injection of THCPSi microparticles into the myocardium were shown to lead to significantly greater activation of inflammatory cytokine and fibrosis promoting genes compared to TOPSi micro and nanoparticles. Neither of the PSi particles altered the cardiac function or the hematological parameters. These data suggests that THCPSi and TOPSi microparticles and TOPSi nanoparticles could effectively improve the cardiac delivery of therapeutic agents, and the PSi biomaterials might serve as a promising platform for the application in the treatment of heart diseases. Acknowledgments We thank Marja Arbelius, Sirpa Rutanen, Kirsi Salo and Mohammad-Ali Shahbazi for expert technical assistance. M.P.A. Ferreira acknowledges a scholarship support from CIMO (grant no. KM-13-8622) and the Drug Research Doctoral Programme of the Faculty of Pharmacy, University of Helsinki. H.A. Santos acknowledges The University of Helsinki Funds and the Academy of Finland (decision no. 252215) and H.J. Ruskoaho acknowledges the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research and the Academy of Finland (decision no. 266661). Tekes Large Strategic Research Opening project no. 40495/13 is also acknowledged for the financial support.

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€ lli MA, et al., In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart, Please cite this article in press as: To Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.078