Bovine serum albumin conjugation on poly(methyl methacrylate) nanoparticles for targeted drug delivery applications

Journal Pre-proof Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for targeted drug delivery applications Camila Guindani, Paulo Emílio Feuser, Arthur Poester Cordeiro, Alessandra Cristina de Meneses, Jonathann Corrêa Possato, Jéssica da Silva Abel, Ricardo Andrez Machado-de-Ávila, Claudia Sayer, Pedro Henrique Hermes de Araújo PII:

S1773-2247(19)31451-0

DOI:

https://doi.org/10.1016/j.jddst.2019.101490

Reference:

JDDST 101490

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 23 September 2019 Revised Date:

2 December 2019

Accepted Date: 28 December 2019

Please cite this article as: C. Guindani, P.E. Feuser, A.P. Cordeiro, A.C. de Meneses, J.C. Possato, J. da Silva Abel, R.A. Machado-de-Ávila, C. Sayer, P.H.H. de Araújo, Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for targeted drug delivery applications, Journal of Drug Delivery Science and Technology, https://doi.org/10.1016/j.jddst.2019.101490. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for future biomedical application

1 2 3 4

Camila Guindani1, Paulo Emílio Feuser1, Arthur Poester Cordeiro1, Alessandra Cristina

5

de Meneses1, Jonathann Corrêa Possato, Jéssica da Silva Abel, Ricardo Andrez

6

Machado-de-Ávila2, Claudia Sayer1, Pedro Henrique Hermes de Araújo1*

7

1

8

Santa Catarina, Brazil

9

2

Department of Chemical Engineering and Food Engineering, Federal University of

Postgraduate Program in Health Science, University of Southern Santa Catarina, Brazil

10 11

*Corresponding author:

12

Pedro Henrique Hermes de Araújo, Prof. Dr.

13

Department of Chemical Engineering and Food Engineering, Federal University of

14

Santa Catarina, 88010 970, Florianópolis - SC, Brazil, phone: 55+ 48 37212533.

15

E-mail: [email protected]

16 17 18

19

GRAPHICAL ABSTRACT

Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for targeted drug delivery applications

1 2 3 4

Camila Guindani1, Paulo Emílio Feuser1, Arthur Poester Cordeiro1, Alessandra Cristina

5

de Meneses1, Jonathann Corrêa Possato, Jéssica da Silva Abel, Ricardo Andrez

6

Machado-de-Ávila2, Claudia Sayer1, Pedro Henrique Hermes de Araújo1*

7 8

1

9

Santa Catarina, Brazil

10

2

Department of Chemical Engineering and Food Engineering, Federal University of

Postgraduate Program in Health Science, University of Southern Santa Catarina, Brazil

11 12

*Corresponding author:

13

Pedro Henrique Hermes de Araújo, Prof. Dr.

14

Department of Chemical Engineering and Food Engineering, Federal University of

15

Santa Catarina, 88010 970, Florianópolis - SC, Brazil, phone: 55+ 48 37212533.

16

E-mail: [email protected]

17 18

ABSTRACT

19

During the application of nanoparticles (NPs) in vivo, it is inevitable that the adsorption

20

of proteins takes place on the surface of the nanocarriers. The formation of this protein

21

corona determines the “identity” of the NPs and how they interact with complex

22

biological media. By controlling the composition of the protein corona it becomes

23

possible to improve properties of NPs and promote targeted drug delivery. In this work,

24

polymethyl methacrylate (PMMA) NPs were conjugated with bovine serum albumin

25

(BSA) by a non-covalent method. The successful conjugation of BSA to PMMA NPs

26

was confirmed by a set of different techniques, such as dynamic light scattering, zeta

27

potential, transmission electron microscopy, Lowry protein quantification assay and

28

flow cytometry. Cytotoxicity assays were also performed and the results shows that NPs

29

and conjugates did not present any cytotoxic effect on the tested cells. Cell uptake

30

assays showed that the conjugation of BSA on PMMA NPs increased cellular uptake by 1

31

HeLa cells in comparison to uncoated PMMA NPs, which is a important feature for

32

successful drug delivery applications. These results are important evidence that it is

33

possible to control the interaction of nanocarriers with cells, by designing a pre-formed

34

protein corona through simple non-covalent conjugation.

35

Keywords:

36

miniemulsion polymerization, cellular uptake, flow cytometry.

bovine

serum

albumin,

polymethyl

methacrylate,

nanoparticles,

37 38

1. INTRODUCTION

39

During the last decade, academia has developed great interest in the application of

40

nanostructures in the biomedical field, giving rise to a new and revolutionary field:

41

nanomedicine [1]. The large surface area to volume ratio, as well as their ability to

42

interact with cells and reach difficult access targets [2], are important features that

43

makes nanomaterials attractive for applications in drug delivery, in vitro diagnostics,

44

biomaterials, active implants and antibiotic materials[3–5]. Besides the possibility of

45

controlling the size of nanoparticles (NPs), engineering its surface is an excellent

46

strategy to tune its interfacial properties and create a wide material platform promoting

47

specific interactions between NPs and biological systems, for different applications [5–

48

7].

49

The exposition of nanoparticle (NPs) to biological fluids allows the adsorption of the

50

proteins present in this environment to the NPs surface, forming a protein corona [8].

51

The presence of the protein corona gives a new identity to the NPs, affecting its

52

interaction with cells and other biomolecules [9,10]. Based in this fact, conjugating

53

NPs with proteins is a way to engineer NPs surface and promote specific interactions

54

between NPs and cells/biomolecules, in order to successfully address delivery-related

2

55

problems and carry drugs to the desired sites of therapeutic action while reducing

56

adverse side effects [11].

57

Effective targeted drug delivery systems relies on the biological interaction between

58

ligands on the surface of NPs and the cell target [12]. Therefore, conjugating NPs with

59

biological ligands is an excellent alternative to promote its binding to specific receptors

60

on the surface of the target cells, and in this way increase cellular uptake of drug-

61

containing NPs, increasing therapeutic efficacy [13].

62

In the present work, polymethyl metacrylate (PMMA) NPs were produced by

63

miniemulsion technique and conjugated with the protein bovine serum albumin (BSA)

64

by a non-covalent method (Scheme 1). PMMA NPs are widely reported in literature as

65

being potential non-toxic nanocarriers for drug delivery for cancer treatment, due to its

66

ability encapsulate anticancer drugs, which are mostly hydrophobic [14–17]. PMMA

67

NPs and conjugates were characterized regarding its size and surface charge. The

68

conjugation of BSA to PMMA NPs was verified by, Lowry protein quantification assay,

69

flow cytometry measurements, and also by transmission electron microscopy (TEM).

70

Finally, biocompatibility, hemocompatibility and cell uptake assays were carried our to

71

evaluate the performance of these NPs as nanocarriers in drug delivery. This is an

72

original work that should contribute to the development of nanotechnology for safer and

73

more efficient health treatments in the future. Scheme 1

74

75

2. MATERIAL AND METHODS

76

2.1 Materials

77

For the synthesis of polymeric NPs, the following reagents were used: methyl

78

methacrylate (MMA), purchased from Arinos Chemistry, azobisisobutyronitrile (AIBN

3

79

98%, Vetec), lecithin (Alpha Aesar) and Crodamol purchased from Alpha Química,

80

Brazil. For conjugation of PMMA NPs, bovine serum albumin (BSA) and fluorescein

81

isothiocyanate (FITC) were purchased from Sigma Aldrich. Distilled water was used

82

throughout the experiments.

83 84

2.2. PMMA NPs synthesis

85

PMMA NPs were obtained by miniemulsion polymerization as described by Feuser et

86

al.[18]. The organic phase containing 2 g of MMA (monomer), 0.09 g of lecithin

87

(surfactant), 0.09 g of Crodamol (co-surfactant) and 0.04 g of azobisisobutyronitrile

88

(AIBN) (initiator). The aqueous phase consisted of distilled water (20 g). The

89

miniemulsion was sonicated for 5 min in an ice bath with amplitude of 70% using a

90

Sonic Dismembrator (Model 500). After miniemulsion preparation, the system was

91

placed in an oil bath at 70 °C and polymerization was carried out for 3 hours.

92

Afterwards, the excess of surfactant present in the miniemulsion was removed by

93

centrifugation/washing cycles. Centrifugation was carried out at 13,000 rpm, for 30

94

min. The supernatant containing the excess of surfactant was removed and the NPs re-

95

dispersed in distilled water (final solid content of 1.6%). Miniemulsions were stored in

96

refrigerator (4 °C) until conjugation and characterization steps were performed. All

97

PMMA NPs characterization assays and in vitro studies were performed after the excess

98

of surfactant present in the miniemulsion was removed.

99

2.2.1. PMMA NPs conjugation with BSA

100

For conjugation of BSA in PMMA NPs, a BSA solution (3 mg.mL-1) was prepared in

101

sodium phosphate buffer 0.1 M, pH 8.0 containing 1mM EDTA. Then, 220 µL of BSA

102

solution was added to 1 mL of PMMA miniemulsion (solid content of 1.6% after

103

surfactant excess removal), and the mixture was incubated overnight in refrigerator. The

4

104

amount of BSA used was established as 10- 8 mol of BSA per mL of miniemulsion,

105

since this amount is theoretically enough to cover the surface of all NPs. After

106

incubation, the miniemulsion containing BSA+PMMA NPs conjugates was purified by

107

performing centrifugation/washing cycles (13,000 rpm for 30 min) for the removal of

108

weakly adsorbed BSA. The supernatant containing free BSA was removed and the

109

conjugates were re-dispersed in distilled water (final solid content of 0.8%). Purified

110

miniemulsions containing the conjugates were stored in refrigerator (4 °C) until

111

characterization steps were performed. All characterization assays and in vitro studies

112

were performed after the purification of BSA+PMMA NPs conjugates.

113 114

2.3. Caracterization

115

2.3.1. Particle size, polydispersity index and surface charge

116

Particle average diameters (Dp) and polydispersity index (PdI) of the uncoated NPs and

117

BSA conjugates were measured by dynamic light scattering (DLS) (Zetasizer Nano S –

118

Malvern). The surface charge of the uncoated NPs and conjugates was investigated by

119

zeta potential measurements (Zetasizer Nano ZS – Malvern). For both analyses, all

120

samples were analyzed in triplicate at room temperature (25 °C), and the average and

121

standard deviation (SD) were calculated.

122

2.3.2. Protein quantification by Lowry assay

123

Lowry assay was performed in order to determine the amount of BSA conjugated to the

124

surface of the nanoparticle. BSA+PMMA NPs conjugates were centrifuged (13,000 rpm

125

for 30 min), and the supernatant containing free BSA was analyzed. The protein content

126

in the supernatant was determined according to the method described by Lowry et

127

al.[19] using Folin’s phenol reagent (phosphomolybdic-phosphotungstic acid reagents).

128

Bovine serum albumin (BSA) was used as a standard. Absorbance was measured at 700

5

129

nm in a SpectraMax spectrophotometer

130 131

2.3.3. Transmission Electron Microscopy (TEM)

132

The visualization of the protein layer surrounding the NPs, as well as the particle

133

morphology and size characterization, was performed by Transmission Electron

134

Microscopy using a JEM-1011 TEM (80 kV), using the negative staining technique.

135

The samples were diluted in distilled water down to 0.5% of solids content, then, one

136

single drop was placed on a 300 mesh carbon-coated copper grid and allowed to dry

137

overnight under room conditions. In sequence, the grid samples were stained with 5%

138

uranyl acetate solution (Riedel-de Haën), and let to dry again at same conditions.

139

2.3.4. Flow cytometry

140

Flow cytometry measurements were performed for PMMA NPs conjugated with BSA,

141

and also for uncoated PMMA NPs. For these measurements, BSA was labeled with

142

fluorescein isothiocyanate (FITC) and then the conjugates were produced in the same

143

way as described in item 2.2.1. Measurements were performed using a BD FACSCanto

144

II Flow Cytometer (laser: 488 nm laser for FITC excitation; emission: 530/30 nm band

145

pass filter). The fluorescent signal was expressed in a histogram and the amount

146

fluorescent positive NPs (%) was determined. Control measurements were performed

147

with pure PBS. BD FACSDivaTM Software v.6.1.3 was used for data acquisition and

148

Flowing Software v.2.5.1 for data analysis.

149

2.4. In vitro studies

150

2.4.1. Cell culture

151

The mouse embryonic fibroblasts (NIH3T3) breast cancer (MDA-MB231) and human

152

cervical cancer (HeLa) cells were grown in Dubelcco’s Modified Eagle's medium

153

(DMEM) (GIBCO, São Paulo, SP, Brazil) supplemented with 10% heat-inactivated

6

154

fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg.mL-1 streptomycin under 5%

155

CO2 at 37 °C.

156

2.4.2. MTT assay

157

For MTT assay, NIH3T3, MDA-MB231 and HeLa cells were seeded at 1×104 cells/well

158

in a 96-well plate and incubated for 24 h at 37 °C. Subsequently, the cells were treated

159

with a medium containing BSA+PMMA NPs at four concentrations: 100, 200, 300 and

160

400 µg.mL-1 and incubated for 24 h. After, the cells were washed two times with PBS

161

(7.4) and the viability was performed by MTT assay. 100 µL of MTT (0.5 µg/mL) were

162

added in each well and the cells were incubated for 3 h. After the incubation period, the

163

MTT was removed and 100 µL of isopropyl alcohol was added to dissolve the formazan

164

crystals. The absorbance was measured at 570 nm using a Loccus LM-96 microplate

165

reader. The experiments were performed in triplicate with three wells for each

166

condition. The results were expressed as the percentage of viable cells in comparison to

167

the control group (untreated cells).

168

2.4.3. Hemolysis assay

169

The human red blood cells (RBCs) from three healthy donors. This study was approved

170

by the medical ethics committee of University of Southern Santa Catarina (Criciúma,

171

Brazil). RBCs were collected in tubes containing 3.2 wt.% of sodium citrate from three

172

volunteers. 4 mL of whole blood was added to 8 mL of a sterile saline solution and the

173

RBCs were isolated from serum by centrifugation at 1500 × g for 5 min. The RBCs

174

were further washed five times with saline solution. Following the last wash, the RBCs

175

were diluted in 2 mL of saline solution and then 70 µL of the diluted RBCs suspension

176

was added to 930 µL of water or saline. The human red blood cells were treated with a

177

medium containing BSA+PMMA NPs at concentrations of 100, 200 and 400µg.mL-1 by

178

gentle stirring at 37 °C for 120 min. After that, the mixture was briefly vortexed and

7

179

centrifuged at 10000 × g for 5 min. 100 µL of supernatant from the sample tube were

180

transferred to a 96-well plate. The absorbance value was measured at 540 nm. As

181

positive and negative controls, 70 µL of the diluted human red blood cells suspension

182

was incubated with 930 µL of distillated water and saline, respectively.

183

2.4.4.Cell Uptake via flow cytometry analysis

184

The cellular uptake of PMMA and BSA+PMMA NPs was analyzed by flow cytometry.

185

For this assay, the fluorescent dye Coumarin-6 was first encapsulated in the NPs, and

186

then the BSA+PMMA NPs conjugates were produced.

187

NIH3T3, MDA-MB231 and HeLa cells were seeded at 2×105 cells/well in a 24-well

188

plate and incubated for 24 h at 37 °C. After incubation period, the cells were incubated

189

for 2 h with 100 µg.mL-1 of PMMA and BSA+PMMA NPs labeled 6-coumarin. After

190

incubation period the cells were washed three times with PBS (pH 7.4) and the cells

191

were collected by trypsinization and washed (PBS) by centrifugation (1 min at 1000

192

rpm). Subsequently the cells (pellets) were re-suspended in PBS for further analysis.

193

Flow cytometry measurements were performed with BD FACSVerse flow cytometer.

194

The fluorescent dye Coumarin-6 was excited with a 488 nm laser. Data analysis was

195

performed using a BD FACSVerse software. Values are expressed as percentage (%) of

196

fluorescent positive cells as an average of at least four independent experiments.

197

2.5. Statistical analysis

198

Data are presented as the mean ± standard deviation (SD) of at least three independent

199

determinations performed in technical triplicate. A One-way ANOVA was used for all

200

experiments at p < 0.05, followed by Bonferroni test as a post-hoc comparison.

201

3. RESULTS AND DISCUSSION

202

3.1. Synthesis of PMMA NPs and conjugation with BSA

8

203

PMMA NPs were successfully synthesized by miniemulsion polymerization method.

204

After the synthesis, NPs were conjugated with the model protein BSA, by the non-

205

covalent method. Particle size, size distribution (PdI) and surface charge of the NPs and

206

conjugates were determined by DLS and zeta potential measurements. The results are

207

shown in Figure 1. For uncoated PMMA NPs, the mean particle diameter was

208

determined to be around 151 ± 1 nm, with a PdI value of 0.22 ± 0.01, typical for PMMA

209

polymerization via miniemulsion. Zeta potential value was -40 ± 2 mV, revealing high

210

miniemulsion stability. After conjugation with BSA, the conjugates kept an uniform

211

size distribution, but the mean particle diameter value increased from 151 ± 1 nm to

212

around 179 ± 2 nm. This increase is probably related to the formation of a BSA layer

213

surrounding the NPs. An increase of around 20 nm in the diameter particle values was

214

also experienced by Simon et al. [20] after conjugation of carboxy- and amino-

215

functionalized polystyrene NPs with IgG depleted plasma. Regarding the surface

216

charge, after conjugation with BSA, the zeta potential value has decreased, reaching a

217

value around -52 ± 1 mV. The decrease in the surface charge value is assigned to the

218

BSA charge contribution, since it has an overall negative charge at pH > 5.5 [21]. This

219

behavior, together with the increase in the mean particle diameter also indicates a

220

successful conjugation of BSA to the surface of the NPs.

221

An estimative of the amount of BSA conjugated to PMMA NPs was obtained by

222

performing the Lowry protein quantification assay. In this assay, the amount of free

223

BSA removed during the purification of the conjugates was determined by the analysis

224

of the supernatant. According to the results obtained by Lowry’s assay, the amount of

225

BSA that remained conjugated to the surface of PMMA NPs was 124 µg of BSA per

226

mL of conjugate’s miniemulsion. This means that the amount of BSA per nanoparticle

9

227

area is 0.0042 BSA molecules.nm-2, which represents around 300 BSA molecules per

228

nanoparticle (approximately 19% of BSA conjugation).

229

Figure 1

230

Flow cytometry measurements were also performed in order to confirm the success of

231

the protein-nanoparticle conjugation. This technique has been used in the field of

232

nanotechnology in order to provide qualitative and semi-quantitative understanding of

233

the nanoparticle bio-interface [22–24]. Through this assay, it is possible to estimate the

234

percentage of NPs that emit fluorescence after being excited by a 488 nm laser. Figure 2

235

shows flow cytometry results for uncoated PMMA NPs and BSA+PMMA NPs. The

236

region evaluated (blue line) was adjusted to the upper right corner, which indicates the

237

presence of conjugates containing BSA-FITC (fluorescently positive events). The large

238

population in the lower left corner is based on non-fluorescent dust/debris, observed on

239

FC results for pure PBS (see supporting information, Figure S1.)

240

Figure 2

241

Flow cytometry results shows that only close to 6% of the uncoated PMMA NPs

242

presented fluorescence, which is expected to this case. For BSA + PMMA NPs

243

conjugates around 38% of the NPs emitted fluorescence, which means the fluorescent

244

BSA was successfully conjugated to PMMA NPs.

245

3.2. Visualization of BSA+PMMA NPs conjugates by Transmission Electron

246

Microscopy

247

Figure 3 presents TEM micrographs of uncoated PMMA (A-C) and BSA+PMMA NPs

248

(1-3) obtained by a negative staining technique with uranyl acetate. This staining

249

method enables the visualization of viruses, bacteria, biological membrane structures,

250

proteins and proteins aggregates, since uranyl acetate scatter electrons strongly and also

251

adsorb to biological matter well [25]. The images confirm the average size of the 10

252

uncoated NPs and conjugates obtained by DLS, as well as its spherical morphology.

253

These images also allow the visualization of a protein layer covering the PMMA NPs

254

(1-3), confirming the successful conjugation of BSA to PMMA NPs. The protein layer

255

appears as small white regions surrounding the nanoparticles, while the background is

256

darker, as a result of the negative staining. For non-conjugated PMMA NPs, these white

257

regions do not appear after negative staining. Similar TEM images of protein-

258

nanostructures conjugates are reported in literature [26–28]. Figure 3

259 260

3.3. Biocompatibility of PMMA nanoparticles and conjugates

261

Biocompatibility assays were performed for BSA+PMMA NPs using non-tumor

262

(NIH3T3 and RBCs) and tumor (MDA-MB231 and HeLa) cells. The biocompatibility

263

of the PMMA NPs used in this work was previously studied by Feuser and collaborators

264

[24], through MTT and hemolysis assays, and the PMMA NPs presented to be

265

biocompatible in all concentrations tested. MTT assays results for BSA+PMMA NPs

266

are presented in Figure 4A, and it shows that the conjugates did not present any

267

cytotoxic effect on NIH3T3, MDA-MB231 and HeLa cells. Hemolysis assay (Figure

268

4B) was also performed in order to evaluate the biocompatibility of new drug delivery

269

systems with RBCs. BSA+PMMA NPs conjugates presented hemocompatibility on

270

RBCs for all tested concentrations. The hemocompatibility is related with the negative

271

charge of BSA+PMMA NPs, which reduces the attraction of erythrocytes onto the NPs

272

surface [29–31]. Figure 4

273 274

3.4. Cell uptake

275

The cellular uptake of PMMA NPs and BSA+PMMA NPs into NIH3T3, MDA-MB231

276

and HeLa cells was evaluated by flow cytometry, after 2h incubation. In spite of serum

11

277

albumin being often reported as a dysopsonin protein [32,33], flow cytometry analysis

278

clearly indicate that the conjugation with BSA increased the NPs internalization (Figure

279

5) by HeLa cells in almost 20%. This behavior was also observed by other authors

280

[34,35], including for application in tumor cells [26,36]. Proteins can undergo

281

conformational changes on its secondary structure while adsorbing onto nanoparticles,

282

being dependent on the size and surface properties of the NPs [37–40]. Since the protein

283

conformation directly influences cell recognition, this might have been a decisive factor

284

in the cellular uptake [35,40,41]. This means that the formulations/methods applied to

285

produce polymeric nanocarriers can directly influence the way that BSA-nanoparticles

286

conjugates interact with cells. Both PMMA NPs and BSA+PMMA NPs conjugates

287

showed higher affinity for cervical cancer cells (HeLa cells) in comparison to the other

288

cell lines tested. These results suggest that the BSA conjugation on PMMA NPs

289

improve the interaction of the nanocarriers with tumor cells and should contribute to

290

increase the efficiency and minimize side effects of future treatments for different types

291

of cancer. Figure 5

292 293

4. CONCLUSIONS

294

In this study, PMMA NPs were prepared by miniemulsion polymerization technique,

295

and afterwards successfully conjugated with the model protein bovine serum albumin

296

(BSA). The successful conjugation of BSA to PMMA NPs was confirmed by multiple

297

characterization methods, such as DLS, zeta potential, Lowry quantification protein

298

assay, and flow cytometry. It was also possible to visualize the conjugates by TEM

299

using a staining technique with uranyl acetate, and the presence of a protein layer

300

attached to the NPs was observed. The biocompatibility of the conjugates was

301

confirmed by MTT and hemolysis assays. Cell uptake assays showed that BSA 12

302

conjugated on PMMA NPs conferred an increase in cellular uptake by HeLa cells,

303

which is a desired feature for drug delivery application in the treatment of tumors. This

304

simple conjugation strategy of engineering the surface of NPs shown to be very

305

promising, and can be applied for the surface modification of PMMA NPs other

306

proteins.

307

SUPPORTING INFORMATION

308

Supporting Information is available online.

309

ACKNOWLEDGEMENTS

310

We would like to thank Denis Dall Agnolo, from the Multi-User Laboratory of Biology

311

Studies (LAMEB/UFSC) for the flow cytometry measurements. We gratefully

312

acknowledge CNPq project number 153829/2018-4 and CAPES (Coordenação de

313

Aperfeiçoamento de Pessoal de Nível Superior) for the financial support.

314

CONFLICTS OF INTEREST

315

The authors declare that they have no conflict of interest.

316

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FIGURE CAPTIONS

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Scheme 1 – (A) Production of PMMA NPs by miniemulsion polymerization technique

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and (B) Conjugation of PMMA NPs with BSA.

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Figure 1 - Particle size distribution obtained by dynamic light scattering: Uncoated

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PMMA and BSA+PMMA NPs conjugates.

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Figure 2 - Flow cytometry analysis of uncoated PMMA NPs, and BSA-FITC + PMMA

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conjugates: (A-B) 2D histogram and (1-2) 1D histogram.

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Figure 3 - TEM images of uncoated PMMA (A-C) and BSA+PMMA NPs (1-3)

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obtained by negative staining technique with uranyl acetate. Red arrows highlight the

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protein layer surrounding BSA+PMMA NPs conjugates.

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Figure 4 - Biocompatibility assay. (A) In vitro cytotoxicity assay of BSA+PMMA NPs

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on NIH3T3, MDA-MB231 and HeLa cells at different concentrations. (B)

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Hemocompatibility assay on human erythrocytes. Positive (C+) and negative (C-)

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control.

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Figure 5 - Quantification (%) of fluorescent positive cells by flow cytometry for PMMA

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NPs and PMMA+BSA NPs conjugates labeled with 6-coumarin after incubation with 20

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NIH3T3, MDA-MB231 and HeLa cells. The NPs were incubated for 2h with 100

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µg.mL-1. *p < 0.05 - Two-way ANOVA followed by the Bonferroni test.

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FIGURES

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Figure 5

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Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for future biomedical application

1 2 3 4

Camila Guindani1, Paulo Emílio Feuser1, Arthur Poester Cordeiro1, Alessandra Cristina

5

de Meneses1, Jonathann Corrêa Possato, Jéssica da Silva Abel, Ricardo Andrez

6

Machado-de-Ávila2, Claudia Sayer1, Pedro Henrique Hermes de Araújo1*

7

1

8

Santa Catarina, Brazil

9

2

Department of Chemical Engineering and Food Engineering, Federal University of

Postgraduate Program in Health Science, University of Southern Santa Catarina, Brazil

10 11

*Corresponding author:

12

Pedro Henrique Hermes de Araújo, Prof. Dr.

13

Department of Chemical Engineering and Food Engineering, Federal University of

14

Santa Catarina, 88010 970, Florianópolis - SC, Brazil, phone: 55+ 48 37212533.

15

E-mail: [email protected]

16 17

HIGHLIGHTS

18 19

•

BSA+PMMA NPs conjugates were successfully produced by a simple noncovalent method;

20 21

•

Flow cytometry provides semi-quantitative understanding of NPs bio-interface;

22

•

Visualization of the protein layer surrounding the NPs was possible by TEM;

23

•

The conjugation of PMMA NPs with BSA increased cell uptake by HeLa cells;

24

•

Production of tailored nanocarriers for application in targeted drug delivery.

25

Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for future biomedical application Camila Guindani1, Paulo Emílio Feuser1, Arthur Poester Cordeiro1, Alessandra Cristina de Meneses1, Jonathann Corrêa Possato, Jéssica da Silva Abel, Ricardo Andrez Machado-deÁvila2, Claudia Sayer1, Pedro Henrique Hermes de Araújo1* 1

Department of Chemical Engineering and Food Engineering, Federal University of Santa

Catarina, Brazil 2

Postgraduate Program in Health Science, University of Southern Santa Catarina, Brazil

*Corresponding author: Pedro Henrique Hermes de Araújo, Prof. Dr. Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina, 88010 970, Florianópolis - SC, Brazil, phone: 55+ 48 37212533. E-mail: [email protected]

AUTHOR STATEMENT

Camila Guindani: Conceptualization, Methodology, Investigation, Validation, Writing Original Draft, Writing - Review & Editing, Visualization, Project administration. Paulo E. Feuser: Conceptualization, Methodology, Investigation, Validation, Writing - Review & Editing. Arthur Poester Cordeiro: Investigation. Alessandra Cristina de Meneses: Investigation. Jonathann Corrêa Possato: Investigation. Jéssica da Silva Abel: Investigation. Ricardo Andrez Machado-de-Ávila: Supervision, Resources, Funding acquisition. Claudia Sayer: Supervision, Resources, Funding acquisition. Pedro Henrique Hermes de Araújo: Conceptualization, Resources, Writing - Review & Editing, Supervision, Funding acquisition.

Florianópolis, September 23th, 2019.

Dear Editor, on behalf of all co-authors, I inform that the manuscript “Bovine serum albumin conjugation on polymethyl metacrylate nanoparticles for targeted drug delivery applications” by Camila Guindani, Paulo Emílio Feuser, Arthur Poester Cordeiro, Alessandra Cristina de Meneses, Jhonathann Corrêa Possato, Jéssica da Silva Abel, Ricardo Andrez Machado-de-Ávila, Cláudia Sayer and Pedro Henrique Hermes Araújo is not biased by any conlicts of interest. Sincerely yours, Prof. Pedro Henrique Hermes de Araújo