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Synthesis, Cellular Uptake, and Biodistribution of Whey-Rich Nanoparticles Xu Zhen, Xin Wang, Chenchen Yang, Qin Liu, Wei Wu, Baorui Liu, Xiqun Jiang*

Whey-poly(acrylic acid) (whey-PAA) nanoparticles are prepared by polymerizing acrylic acid (AA) monomer in the presence of whey protein in a complete aqueous medium. The properties, drug loading, and release as well as in vitro cytotoxicity of whey-PAA nanoparticles are examined. The cellular uptakes and penetration of nanoparticle in the SH-SY5Y monolayer cells and multicellular tumor spheroids are observed. The in vivo distribution of the nanoparticles in tumor-bearing mice is evaluated. Confocal laser scanning microscopy and colocalization images show that the nanoparticles are well internalized by the cells through the endocytosis mechanism. Drug-loaded whey-PAA nanoparticles can penetrate multicellular tumor spheroids more deeply. In vivo nearinfrared fluorescence imaging examination and in vivo DOX distribution show that the drugloaded whey-PAA nanoparticles can well accumulated in the tumor site. Thus, these whey-rich nanoparticles seem to be very promising drug carriers for drug delivery.

Recently, protein-based nanoparticles have attracted increasing attention due to their excellent biocompatible and biodegradable properties. The nanoparticles based on proteins such as silk protein, gelatin, casein and albumin have been used as drug delivery systems for cancer treatments.[1–4] However, the utilization of native proteins for drug delivery systems is still limited, and the inherent

X. Zhen, X. Wang, C. Yang, Dr. W. Wu, Prof. X. Jiang Department of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory for Nanotechnology, Nanjing University, Nanjing 210093, P. R. China E-mail: [email protected] Dr. Q. Liu, Prof. B. Liu Department of Oncology, Affiliated Drum Tower Hospital, Medical College of Nanjing University, Nanjing 210093, P. R. China

ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

features of proteins in biological performance have not been fully exploited yet. Milk proteins are recognized to have a wide range of nutritional, functional and biological activities. The distinct physiological functions in vivo of caseins and whey proteins have been demonstrated.[5] Today, milk protein, particularly whey protein have found extensive use in dietary supplements, and clinical and sports diet formulations.[6] Whey proteins are comprised of b-lactoglobulin (b-lg), a-lactalbumin (a-la), bovine serum albumin (BSA) and Immunoglobulins (lg).[7] Among them, 70% of all whey proteins are b-lg and a-la. b-lg has 162 amino acid residues, and a molecular weight of 18.3 kDa, while a-la has 123 amino acid residues, and a molecular weight of 14.4 kDa. So far, some efforts have been made for the preparation of whey protein nanoparticles. For example, whey protein nanoparticles were prepared by pH-cycling treatment, and desolvation with ethanol.[8–10] In these investigations, the physical properties of whey protein nanoparticles such as

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1. Introduction

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swelling, turbidity, and viscosity were characterized. However, the biological behaviors and drug delivery of whey protein nanoparticles have not been explored and known little. In present work, we report the preparation of whey protein-poly(acrylic acid)(whey-PAA) nanoparticles through a biopolymer-monomer pair approach.[2,11] The wheyPAA nanoparticles were spontaneously formed by polymerizing acrylic acid (AA) monomer in the presence of whey protein in a complete aqueous medium. The size of nanoparticles can be controlled by changing reaction conditions. The drug loading and release of whey-PAA nanoparticles were evaluated using doxorubicin (DOX) as a model drugs. Further, we investigate the internalization of the whey-PAA nanoparticles in two-dimensional (2D) monolayer cells and three-dimensional (3D) multicellular tumor spheroids (MCTS).The penetration of free DOX and DOX-loaded whey-PAA nanoparticles in MCTS was compared. Finally, the in vivo behaviors of DOX-loaded whey-PAA nanoparticles in tumor-bearing mice were examined based on near-infrared fluorescence (NIRF) imaging and in vivo DOX ditribution.

2. Experimental Section

paper to remove any aggregation. The resultant suspension was cooled at room temperature and dialyzed against the aqueous medium with pH 3.5 for 24 h using a dialysis bag with a cut-off molecular weight of 14 kDa to remove residual monomers and other small molecules. To improve the stability of whey-PAA nanoparticles, the wheyPAA nanoparticles were cross-linked in the aqueous solution through adding the desired amount of 2,20 -(ethylenedioxy)bis(ethylamine) in the presence of EDC at room temperature for 12 h. The cross-linked whey-PAA nanoparticles were dialyzed against deionized water (pH 7.4) for 24 h using a dialysis bag with a cut-off molecular weight of 14 kDa to remove the unreacted crosslinkers.

2.3. Characterization of the Whey-PAA Nanoparticles The mean hydrodynamic diameter and size distribution of nanoparticles were evaluated by dynamic light scattering (DLS) using a Brookheaven BI9000AT system (Brookheaven Instruments Corporation, USA). All DLS measurements were done with a laser wavelength of 660.0 nm at 25 8C, and each batch was analyzed in triplicate. The morphology of whey-PAA nanoparticles was observed by transmission electron microscopy (TEM, JEOLTEM100, Japan). One drop of the nanoparticles suspension was placed on a 200-mesh nitrocellulose-covered copper grid. The grid was allowed to dry at room temperature without staining, and was examined with the TEM.

2.1. Materials Whey protein was purchased from Sigma–Aldrich. The isoelectric point of whey protein is about 4.8. Acrylic acid (AA) was purchased from Nanjing Chemical Reagent Co. Ltd. 4,40 -azobis(4-cyanovaleric acid), 2,20 -(ethylenedioxy)bis(ethylamine), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC), Rhodamine B isothiocyanate (RBITC), Lyso-Tracker Red and NIR-797-isothiocyanate were obtained from Sigma Chemical Co. Doxorubicin hydrochloride (DOX) was obtained from Shenzhen Main Luck Pharmaceuticals Inc. (Shenzhen, China). All other reagents were of analytical grade and used without further purification. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) was purchased from BDH Laboratory Supplies (Poole, Dorset, England). Murine hepatic H22 cell line and human neuroblastom cancer cell line SH-SY5Y were obtained from Shanghai Institute of Cell Biology (Shanghai, China). Male ICR mice (6–8 weeks old and weighing 18–22 g) were purchased from Animal Center of DrumTower Hospital (Nanjing, China).

2.2. Preparation of Whey-PAA Nanoparticles Whey protein (50 mg) was dispersed in 10 mL of acrylic acid (AA, 100 mL) aqueous solution, and a predetermined amount of initiator 4,40 -azobis(4-cyanovaleric acid) was added into the solution. The polymerization of AA monomer was initiated at 90 8C under argon atmosphere. As the opalescence appeared in the reaction system, which was a signature of the formation of whey-poly(acrylic acid) (whey-PAA) nanoparticles, the reaction was allowed to proceed for another 3 min at 90 8C. The suspension was then filtered with filter

2.4. Preparation of DOX-Loaded Whey-PAA Nanoparticles The DOX-loaded nanoparticles were prepared by mixing a certain amount of DOX with 4 mL of aqueous medium of whey-PAA nanoparticles. The mixture was stirred at room temperature for 24 h in the dark to allow the nanoparticles to load DOX sufficiently. Then the mixture was centrifuged at 14 000 rpm for 30 min to separate free DOX. The amount of free DOX in the supernatant was quantified by measuring the absorbance at 480 nm by UV–Vis spectrophotometer, and the mass of the DOX-loaded nanoparticles was weighed after being dried under vacuum overnight. The drug loading efficiency (LE) and loading content (LC) of the nanoparticles (NPs) were calculated as follows: LE ð%Þ ¼

weigh of DOX in NPs  100% weight of the feeding drug

ð1Þ

LC ð%Þ ¼

weigh of DOX in NPs  100% weight of the DOX  loaded NPs

ð2Þ

2.5. Kinetic Stability of DOX-Loaded Whey-PAA Nanoparticles The kinetic stability of DOX-loaded whey-PAA nanoparticles was observed by measuring the particle size as a function of time. The samples were dissolved in H2O, saline, and PBS with pH of 7.4 at 37 8C and evaluated by DLS with a Brookheaven BI9000AT system.

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At various time points, the hydrodynamic diameters and the scattering light intensity were measured by DLS at 25 8C.

2.6. In Vitro Drug Release

further incubation at 37 8C or 4 8C, the cover glass containing adherent cells was taken out, washed three times with PBS to remove free RBITC-labeled whey-PAA nanoparticles and fixed through inversely putting the cover glass onto the glass slide. Cells were observed by confocal laser scanning microscopy (CLSM, Zeiss LSM 710, Germany) at an excitation wavelength of 543 nm.

A certain amount of DOX-loaded nanoparticles were suspended in 1 mL deionized water. Then the solution was placed into a dialysis bag (14 kDa cut-off) and immersed into 5 mL of 0.01 M phosphate buffered saline (PBS). This release system was adjusted with PBS to pH 5.0, 6.0 and 7.4, at 37 8C, respectively, and gentle agitation in the dark. Periodically, 5 mL of the release medium was withdrawn and then 5 mL of fresh PBS was added to the system. DOX concentration in the sampled medium was determined by a fluorescence spectrometer (RF-5301 PC, SHIMADZU, Japan) at an excitation wavelength of 480 nm and an emission wavelength of 560 nm. The cumulative amount of DOX released from the nanoparticles was plotted against time.

SH-SY5Y cells were first incubated with 100 nM Lyso-Tracker Red for 30 min at 37 8C in a humidified atmosphere of 5% CO2, and washed with culture medium. FITC-labeled nanoparticles (200 mL) was subsequently added into the cell culture medium. After incubation for 4 h at 37 8C, the cells were washed three times with PBS. DAPI was employed to dye the nucleus zone of the cells. Then the cells were observed with CLSM. Lyso-Tracker Red excitation was achieved with a 543 nm HeNe laser.

2.7. In Vitro Cytotoxicity

2.10. Cellular Uptake and Penetration in MCTS

Cytotoxicity of nanoparticles against human derived neuroblastoma cell line (SH-SY5Y cells) was assessed by MTT assay as previously described.[12] Briefly, SH-SY5Y cells were seeded on 96-well plates with a density around 5 000 cells per well and allowed to adhere for 24 h prior to the assay. The cells were co-incubated with a series of doses of free DOX, DOX-loaded nanoparticles at 37 8C for 48 h. Then, 50 mL of MTT indicator dye (5 mg mL1 in PBS, pH 7.4) was added to each well, and the cells were incubated for another 2 h at 37 8C in the dark. The medium was withdrawn and 200 mL acidified isopropanol (0.33 v/v HCl in isopropanol) was added in each well and agitated thoroughly to dissolve the formazan crystals. The solution was transferred to 96-well plates and immediately monitored on a microplate reader (Bio-Rad, Hercules, CA, USA.). Absorption was measured at a wavelength of 490 and 620 nm as a reference wavelength. The values obtained were expressed as a percentage of the control cells to which no nanoparticles were added. The cytotoxicity of empty whey-PAA nanoparticles was also evaluated as described above.

The multicellular tumor spheroids (MCTS) were produced as described in previous work.[12] SH-SY5Y multicellular tumor spheroids with diameters between 200 and 300 mm were harvested after approximately 7–9 days of growth. For the experiment, about 20 spheroids were handpicked with a Pasteur pipette and transferred to a 5 mL eppendorf tube. Appropriate concentrations of RBITC-labeled nanoparticles were then added to the spheroids suspension and co-cultured at 37 8C and 4 8C for 24 h. The medium was then removed and spheroids were washed with PBS (pH 7.4) before observation. The cellular uptake of nanoparticles in MCTS was observed by CLSM. For the penetration examination of the nanoparticles in MCTS, individual spheroids were imaged every 5 mm section from the bottom to top and the penetration of free DOX and DOX-loaded nanoparticles in MCTS was observed by CLSM.

2.8. In Vitro Cellular Uptake The whey-PAA nanoparticles were labeled with rhodamine B isothiocyanate (RBITC). Briefly, 1 mL of anhydrous DMSO containing 1 mg RBITC was added into 4 mL whey-PAA nanoparticles solution, and the mixture was stirred for 24 h at room temperature in the dark. Then, the RBITC-labeled whey-PAA nanoparticles were separated by centrifugation and unreacted RBITC was removed. Finally, the obtained RBITC-labeled nanoparticles were dispersed in aqueous solution for in vitro cellular uptake. The SH-SY5Y cells were seeded at a density of 1  106 cells per well in a six-well plate containing a cover glass and allowed to adhere for 24 h in a humidified atmosphere of 5% CO2 at 37 8C to achieve a confluence of approximately 80%. After 24 h incubation, the medium was replaced by fresh temperature-equilibrated complete medium, and a determined amount of the RBITC-labeled whey-PAPBA nanoparticles were added into the plates. With 4 h

2.11. Real-Time NIRF Imaging Examination In Vivo NIR-797-isothiocyanate was used to label DOX-loaded whey-PAA nanoparticles. Briefly, 2 mg of NIR-797-isothiocyanate was dissolved in 1 mL of DMSO, then 200 mL of the dye solution was added into 4 mL of nanoparticle solution and the mixing solution was allowed to shake gently at 37 8C for 12 h. Unconjugated NIR-797 was removed by ultrafiltration (MWCO, 14 kDa) for 24 h. All animal studies were performed in compliance with guidelines set by the Animal Care Committee of Nanjing University. H22 tumor cells (2–3  106 cells per mouse) were inoculated subcutaneously to the ICR mice at the left flank. The NIR-797 labeled nanoparticles were intravenously administrated into H22 tumor bearing mice after the tumor was established. After i.v. administration, the timedependent biodistribution in tumor bearing mice was imaged using the Maestro EX fluorescence imaging system (Cambridge Research & Instrumentation, CRi, USA). The mice were anesthetized and placed on an animal plate heated to 37 8C. Light with a central wavelength at 704 nm was selected as the excitation source. In vivo spectral imaging from 740 to 950 nm (10 nm step) was carried out.

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2.9. Co-Localization of Nanoparticles and Cells

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Scans were conducted at 1, 3, 6, 12, 24, 48, 72, 96 h, 120, and 144 h post administration.

2.12. In Vivo DOX Accumulation ICR mice were inoculated on the left flank with H22 tumor cells (4–6  106 cells per mouse). The free DOX and DOX-loaded wheyPAA nanoparticles were administered intravenously at a dose of 6 mg kg1 on a DOX basis 7 days after the inoculation. The mice were sacrificed at 1, 3, 6, 12, 24, 48, and 72 h after administration (n ¼ 3 at each time point). Subsequently, the blood was collected and plasma was obtained by centrifuging whole blood at 13 000 rpm for 10 min. The tumor and heart were excised and weighed. These tissues and plasma were suspended in 70% ethanol with 0.3 N HCl and intensely homogenated. After a further centrifugation, DOX fluorescence intensity in the supernatant was measured with a fluorescence spectrometer at an excitation wavelength of 480 nm and emission wavelength of 560 nm. The DOX concentrations in blood and each tissue sample were determined according to standard curves. The measured average values of each tissue from the blank group were served as background and deducted from corresponding sample of the administrated mice.

2.13. Statistical Analysis Quantitative data were expressed as mean  SD. Statistical comparisons were made by ANOVA analysis and Student’s t-test.

3. Results and Discussion Figure 1. DLS and TEM images of whey-PAA nanoparticles with the size of A) 163 nm, B) 203 nm, and C) 246 nm.Scale bar in TEM images is 200 nm.

3.1. Preparation of Whey-PAA Nanoparticles In our previous work,[2,11] we have developed a facile strategy to prepare polymer nanoparticles in a reaction system consisting of a biopolymer and a polymerizable monomer. In this study, we chose whey protein as the biopolymer and acrylic acid (AA) as the polymerizable monomer. The whey-PAA nanoparticles were synthesized by polymerizing AA monomers in the presence of whey protein in complete aqueous solution. With the polymerization of the AA monomers, the electrostatic interaction between the positively charged amino groups of whey and the negatively charged carboxyl groups of poly(acrylic acid)

turns into a stronger one. When polymerization reaches a certain degree, the stable whey-PAA nanoparticles are spontaneously formed. Further, the whey-PAA nanoparticles were cross-linked using 2, 20 -(ethylenedioxy)bis(ethylamine) in the presence of EDC to obtain more stable structure. The size and size distribution of the nanoparticles formed weremeasured by DLS, and shown in Figure 1 and Table 1. We find that these nanoparticles exhibit a unimodal

Table 1. Basic Properties of whey-PAA nanoparticles.

Whey [mg] 50

a)

AA [mL]

Temperature [8C]

Initiator [mg]

Diametera) [nm]/PDIb)

z-Potentialc) [mV]

100

90

3

163/0.12

23.21  1.4

80

100

90

3

203/0.11

18.76  0.8

100

100

90

3

246/0.13

21.26  1.9

Measured by DLS; b)PDI ¼ Polydispersity index; c)pH 7.4 after cross-linking.

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size distribution, and the hydrodynamic diameter of wheyPAA nanoparticles can be controlled in the range of about 160–240 nm by changing the feeding ratio of raw materials, as shown in Table 1. As the amount of whey protein added increases from 50 to 100 mg in the reaction system, the diameter of whey-PAA nanoparticles obtained increases from 160 to 240 nm as the other conditions keep constant. In addition, all these nanoparticles maintain a narrow size distribution (polydispersity < 0.15). From Zeta potential measurement, it is found that these whey-PAA nanoparticles have the negative surface potential in pH 7.4. Figure 1 also shows TEM images of whey-PAA nanoparticles. It can be seen that these nanoparticles have a spherical morphology and TEM observations give a smaller particle size than DLS measurements due to the dry state in TEM observation. Thus, whey-PAA nanoparticles are successfully prepared by polymerizing AA monomer in the presence of whey protein. In following experiments, the whey-PAA nanoparticles with the hydrodynamic diameter of 163 nm were selected due to a relatively small diameter. 3.2. DOX Loading and In Vitro Release Considering that whey-PAA nanoparticles contain many carboxyl groups in the whey and PAA molecules, they seem

to be very appropriate to be the carriers for DOX since the DOX has an amino group in its structure. DOX-loaded whey-PAA nanoparticles were prepared by an incubation method exploiting the electrostatic interactions between the protonated amino groups of DOX and carboxyl groups in whey-PAA nanoparticles. After DOX loading, the average diameter of DOX-loaded nanoparticles increases from 163 nm to 176 nm measured by DLS. The drug loading efficiency of DOX in the nanoparticles is 96% and the drug loading content of the nanoparticles reaches 23%. The large amount of carboxylic groups in whey-PAA nanoparticles should be responsible for such high drug loading efficiency and the loading content. Since some types of colloids undergo considerable agglomeration in saline and biological media with potential interference in extravasation at the tumor site,[13] the kinetic stability of DOX-loaded whey-PAA nanoparticles was examined by DLS. The time-dependent changes in the light scattering intensity and the average hydrodynamic diameter of the nanoparticles in H2O, saline and PBS with pH 7.4 at 37 8C are shown in Figure 2A–C. It can be seen that in all cases, only a slightly decrease in the relative light scattering intensity is observed as time elapses and the size of the DOX-loaded whey-PAA nanoparticles is almost invariant even after 6-day store. These results suggest that

Figure 2. The stability of DOX-loaded whey-PAA nanoparticles in A) H2O A), B) saline, and C) PBS with pH 7.4. D) In vitro release profiles of DOX from nanoparticles in PBS (pH 5.0, 6.0, 7.4, 0.01 M) at 37 8C.

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DOX-loaded whey-PAA nanoparticles are kinetically stable at least in 144 h period in H2O, saline and PBS, and appropriate for further in vivo systemic drug delivery. Next the release profiles of DOX from the nanoparticles were traced using a fluorescence spectrometer. Figure 2D shows the in vitro release of DOX from nanoparticles in PBS with different pH values. It can be seen that in vitro DOX release from the nanoparticles is pH-dependent and exhibits a steady continued release pattern. Only 6% of the encapsulated DOX is released from the nanoparticles in PBS within 144 h at pH 7.4, while the release amount of DOX increase dramatically to 24 and 74% at pH 6.0 and 5.0, respectively. This result suggests that the DOX-loaded nanoparticles are highly stable in the blood and neutral environment, and the release of DOX speeds up in low pH environment. This is primarily ascribed to the weakening of electrostatic interaction between the protonated amino groups of DOX and carboxyl groups in whey-PAA nanoparticles at low pH environments. Such pH-dependent release behavior is desirableas the ideal drug release should be slow in the neutral environment of systemic circulation in vivo and fast in the acidic environment of tumor (pH 6.0– 6.5) and intracellular sites (pH 4.5–5.5 in lysosomes, pH 5.5– 6.0 in endosomes).[14–16]

3.3. In Vi tro Cytotoxicity The cytotoxicity of the nanoparticles was evaluated by the metabolic viability of human neuroblastoma cell line SH-SY5Y cells using MTT assay. The high cell viability of empty whey-PAA nanoparticles indicates that the empty whey-PAA nanoparticles are non-toxic at all test concentrations (Figure 3A). On the other hand, a dose-dependent cytotoxicity is observed for free DOX and DOX-loaded whey-PAA nanoparticles (Figure 3B). The cytotoxicity of DOX-loaded whey-PAA nanoparticles is slightly lower than that of free DOX in the tested concentrations. Considering that only 45% of the loaded DOX is released from the nanoparticles within 48 h at pH 5.0, the cytotoxicity of DOX-loaded whey-PAA nanoparticles is comparatively high, which may associate with the cell uptake ability of whey-PAA nanoparticles.

3.4. Cellular Uptake As the drug nanocarriers, the internalization of the nanocarriers into cells is the prerequisite for both in vitro and in vivo drug delivery. To examine the cellular uptake of the whey-PAA nanoparticles, SH-SY5Y cells were incubated with RBITC-labeled whey-PAA nanoparticles at 37 and 4 8C for 4 h, and then observed by CLSM. Figure 4A shows typical CLSM image of SH-SY5Y cells after incubation with wheyPAA nanoparticles at 37 8C. It can be seen that a large

Figure 3. A) In vitro cytotoxicity of empty whey-PAA nanoparticles against SH-SY5Y cells after co-incubation for 24 h (concentration based on whey-PAA nanoparticles). B) In vitro cytotoxicity of free DOX and DOX-loaded whey-PAA nanoparticles against SH-SY5Y cells after co-incubation for 48 h (concentration based on DOX).

number of whey-PAA nanoparticles (red color) are internalized in the cells, and exhibit a diffused distribution of red color in the cytoplasm region. Nevertheless, no whey-PAA nanoparticle is found in the nucleus region, which is stained into blue by DAPI, suggesting that the whey-PAA nanoparticles are unable to penetrate the cell nucleus. To figure out the cellular uptake mechanism of the whey-PAA nanoparticles, SH-SY5Y cells were incubated with RBITClabeled whey-PAA nanoparticles at 4 8C for 4 h. Because the lipid bilayer of the cell would be hardened at low temperature, the endocytosis process would be substantially weakened by lowering temperature.[17] Indeed, the significantly weak red fluorescence inside the cell cytoplasm is observed for the cells incubated with whey-PAA nanoparticles at 4 8C (Figure 4B), suggesting that the cellular uptake of whey-PAA nanoparticles follows the endocytosis mechanism since the endocytosis restricted at 4 8C. A semiquantitative analysis of mean fluorescence intensity of

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Figure 4. CLSM images of SH-SY5Y cells after 4 h incubation with whey-PAA nanoparticles labeled by Rhodamine B. A) at 37 8C and B) at 4 8C. C) Mean fluorescence intensity in cells for RBITC-labeled whey-PAA nanoparticles at 37 8C and 4 8C. Scale bar ¼ 50 mm.

RBITC-labeled whey-PAA nanoparticles inside cells is displayed in Figure 4C. Quantitatively, the mean fluorescence intensity of the cells at 37 8C is about ninefold higher than that at 4 8C (17 vs 1.9). This result verifies again that the cellular uptake of whey-PAA nanoparticles follows the endocytosis mechanism. To investigate the intracellular distribution of whey-PAA nanoparticles, Lyso Tracker (red) was used as an endsomal/ lysosomal marker, while the nanoparticles were labeled by FITC (green). After 4 h of incubation, the red punctate fluorescence signals from the marker are visible in the cytosol (Figure 5), indicating that the marker can well point the endosomal/lysosomal positions out. As shown in

Figure 5. CLSM images of SH-SY5Y cells incubated with FITC labeled whey-PAA nanoparticles (green). Endosomes/lysosomes of the cells are marked by Lyso Tracker (red) and the nuclear were stained with DAPI (blue). Scale bar ¼ 50 mm.

3.5. Uptake and Penetration in Multicellular Spheroids To further investigate the cellular uptake and penetration behaviors of whey-PAA nanoparticles in vitro, SH-SY5Y cell line was used to culture the 3D tissue model called multicellular tumor spheroids (MCTS). Compared to 2D cell culture, 3D cell culture is more correlative with in vivo solid tumors in the 3D network of the cell-cell and cellextracellular matrix (ECM) interactions.[18–20] Also, the tissue penetration behavior of nanoparticles in vitro can be examined using MCTS.[21,22] The RBITC-labeled whey-PAA nanoparticles were incubated with the MCTS for 24 h at 37 8C and 4 8C, respectively. Similar to in monolayer cultures, the whey-PAA nanoparticles are strongly internalized in the MCTS at 37 8C (Figure 6A). The spheroids exhibit a diffused distribution of red color in the peripheral and deeper region after incubation. Also, it can be seen that the whey-PAA nanoparticles localize 40–50 mm deep in the MCTS after 24 h of incubation at 37 8C, as shown in Figure 6A. This result suggests that the whey-PAA nanoparticles are able to not only penetrate cell membrane barriers but also transport to the deeper regions of the MCTS. Not surprisingly, there are few whey-PAA nanoparticles inside the MCTS at 4 8C (Figure 6B). The semiquantitative analysis also demonstrates that the mean fluorescence intensity of the cells at 37 8C is about sevenfold higher than that at 4 8C (36 vs 5), being consistent with the result of 2D cellular uptake. Next we examine the transport behavior of DOX in MCTS utilizing the fluorescence of DOX itself. After incubation for

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Figure 5, the co-localization (yellow) of Lyso Tracker (red) with FITC-labeled nanoparticles (green) demonstrates that the majority of nanoparticles are localized in endosomes or lysosomes, and the minority nanoparticles are in cytosol after 4 h of incubation. This result indicates again that the endocytosis is the most likely pathway of cellular uptake of whey-PAA nanoparticles.

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Figure 6. CLSM images of MCTS incubated with RBITC-labeled whey-PAA nanoparticles A) at 37 8C and B) at 4 8C. C) Mean fluorescence intensity in MCTS for RBITC-labeled whey-PAA nanoparticles at 37 8C and 4 8C. Scale bar ¼ 100 mm.

24 h at 37 8C, with free DOX and DOX-loaded whey-PAA nanoparticles (200 mg based on DOX), respectively, MCTS were imaged every 5 mm section from the bottom to top (Figure 7). Interestingly, DOX-loaded whey-PAA nanoparticles clearly penetrated deeper in the MCTS compared to free DOX. From Figure 7B and C, it can be seen that the fluorescence intensity of free DOX decreases from the edge of the equatorial section to the center of MCTS (Figure 7A). Compared to the periphery, the central part of MCTS has the negligible fluorescence intensity (Figure 7B), indicating

limited penetration of free DOX in MCTS. Similar result was also observed in other work.[18] On the other hand, it is noteworthy that DOX-loaded whey-PAA nanoparticles penetrate into the MCTS deeply after incubation with MCTS for 24 h at 37 8C (Figure 7D and E). The fluorescence distribution diagram shows that a lot of DOX from the nanoparticles accumulate in the peripheral region with the depth about 50 mm (Figure 7E), and the fluorescence intensity of the central part of MCTS is substantial (Figure 7F). These results suggest that DOX-loaded whey-

Figure 7. CLSM section images of MCTS after incubated with A) free DOX and D) DOX-loaded whey-PAA nanoparticles for 24 h (concentration of 200 mg based on DOX). The images were taken every 5 mm section from the top to bottom. B,E) The representative middle region images of MCTS in (A,D), respectively. Scale bar ¼ 100 mm. Fluorescence intensity distribution along a horizontal line across the optical section of the equatorial plane (middle) of MCTS after incubation with C) free DOX and F) DOX-loaded whey-PAA nanoparticles.

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PAA nanoparticles can enter into the MCTS and then DOX is released from the nanoparticles to penetrate the MCTS more deeply, while free DOX only distribute in the periphery. The better penetration of DOX from the nanoparticles than free DOX in MCTS is probably due to good penetration of whey-PAA nanoparticles themselves and the acidic environment in deep region of MCTS which causes fast release of DOX from the nanoparticles.[23,24] 3.6. Real-Time NIRF Imaging To investigate the fate of DOX-loaded whey-PAA nanoparticles in a living body, the non-invasive NIRF imaging technique was used to visualize the tissue distribution of the nanoparticles in vivo. The DOX-loaded whey-PAA nanoparticles were labeled with a NIRF dye, NIR-797, and then injected into subcutaneous hepatic H22 tumor-bearing mice via the tail vein. Figure 8 displays the in vivo NIR fluorescence images at different time after injection. The different fluorescence intensities are represented by different colors as shown in color histogram. From Figure 8, , it can be seen that the nanoparticles mainly accumulate in liver and intestine in the initial 3 h, indicating that some of the nanoparticles are rapidly eliminated from the circulation and cleared by hepatobiliary excretion process. After 3 h, the fluorescence signal of whey-PAA nanoparticles appears in tumor site and becomes stronger and stronger as time escapes. After 12 h, the fluorescence intensity in liver tissue starts to decrease while fluorescence intensity in the

tumor region still increases. At 72 h post-injection, the fluorescence intensity in tumor site is already stronger than that in liver. At 144 h post-injection, the whey-PAA nanoparticles left in liver is much less than before while the fluorescence signal in the tumor site is still very strong. These results suggest that a portion of the whey-PAA nanoparticles have the ability to escape the recognition by the phagocytic cells and the reticuloendothelial system (RES), leading to a long blood circulation time and prominent EPR effect. 3.7. In Vivo DOX Distribution The in vivo DOX distributions of DOX-loaded whey-PAA nanoparticles and free DOX in H22 tumor-bearing mice were evaluated after i.v. injection at a DOX dose of 6 mg kg1. Figure 9 shows the distribution of DOX at different time points. It is noteworthy that the DOX concentration in blood for whey-PAA nanoparticles formulation is about fourfold higher than that of free DOX at the same sampling time. The half-life (t1/2) of DOX in blood circulation is calculated to be 6.1 h for whey-PAA nanoparticles, which is threefold longer than free DOX (t1/2 ¼ 2.1 h), demonstrating that the decay of DOX concentration in blood for the free drug is very rapid and nanoparticles as DOX carriers can significantly prolong the circulation time of DOX in the blood. For DOX accumulation in tumor, the concentration of free DOX reaches its maximum (3.5% ID g1 tissue) at 1 h after

Figure 8. The NIRF images of H22 tumor-bearing mice following i.v. injection of NIR-797 labeled DOX-loaded whey-PAA nanoparticles. Tumor areas were surrounded with dotted lines. Macromol. Biosci. 2014, DOI: 10.1002/mabi.201400018 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 9. Biodistribution of DOX-loaded whey-PAA nanoparticles and free DOX.

injection, and then the concentration starts to decrease until the end of the experiment. However, the DOX concentration in tumor for the whey-PAA nanoparticles keeps increasing from 1 h after injection and reaches its maximum (6.2% ID g1 tissue) at 12 h post-injection. At 72 h, the DOX concentration in tumor for the whey-PAA nanoparticles is still much higher (4.5% ID g1 tissue) than that of free DOX (1.0% ID g1 tissue). This result suggests that the whey-PAA nanoparticles can make DOX stay in the tumor for a longer action time with relatively higher DOX concentration due to a passive targeting against solid tumors by EPR effect. In addition, it is found that the DOX concentration in heart for free DOX at 1 h post-injection is fivefold higher than DOX-loaded whey-PAA nanoparticles (15.7 vs 2.9% ID g1 tissue). This result indicates that DOXloaded whey-PAA nanoparticles can remarkably reduce the cardiotoxicity of DOX which is the main side effect caused by free DOX.

spherical shape and exhibited a satisfactory kinetic stability. In vitro cytotoxicity revealed that a dosedependent cytotoxicity is observed for free DOX and DOX-loaded whey-PAA nanoparticles. The monolayer cells and MCTS uptake of whey-PAA nanoparticles demonstrated that the internalization of nanoparticles followsthe endocytosis mechanism. DOX-loaded whey-PAA nanoparticles could penetrate MCTS more deeply, while free DOX only distribute in the periphery. In vivo NIRF imaging and in vivo DOX distribution demonstrated that the DOXloaded whey-PAA nanoparticles had prominent tumor targeting ability due to the long blood circulation time and EPR effect. These properties of drug-loaded whey-PAA nanoparticles suggest that the whey-PAA nanoparticles are suitable as a drug delivery system. Acknowledgements: This work was supported by the National Natural Science Foundation of China (Grant No. 51033002 and 51273090), and Program for Changjiang Scholars and Innovative Research Team in University.

4. Conclusion In this study, whey-PAA nanoparticles with the tunable size were prepared. The whey-PAA nanoparticles showed

Received: January 13, 2014; Revised: March 21, 2014; Published online: DOI: 10.1002/mabi.201400018

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Keywords: cellular uptake; drug delivery; protein nanoparticles; whey

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Synthesis, cellular uptake, and biodistribution of whey-rich nanoparticles.

Whey-poly(acrylic acid) (whey-PAA) nanoparticles are prepared by polymerizing acrylic acid (AA) monomer in the presence of whey protein in a complete ...
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