Herceptin conjugated PLGA-PHis-PEG pH sensitive nanoparticles for targeted and controlled drug delivery

International Journal of Pharmaceutics 487 (2015) 81–90

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Herceptin conjugated PLGA-PHis-PEG pH sensitive nanoparticles for targeted and controlled drug delivery Zilan Zhou a , Apurva Badkas a , Max Stevenson b , Joo-Youp Lee a, * , Yuet-Kin Leung b a Chemical Engineering Program, Department of Biomedical, Environmental, and Chemical Engineering, University of Cincinnati, Cincinnati, OH 45221-0012, USA b Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, OH 45221-0012, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 November 2014 Received in revised form 11 March 2015 Accepted 27 March 2015 Available online 9 April 2015

A dual functional nano-scaled drug carrier, comprising of a targeting ligand and pH sensitivity, has been made in order to increase the specificity and efficacy of the drug delivery system. The nanoparticles are made of a tri-block copolymer, poly(D,L lactide-co-glycolide) (PLGA)-b-poly(L-histidine) (PHis)-b-polyethylene glycol (PEG), via nano-precipitation. To provide the nanoparticle feature of endolysosomal escape and pH sensitivity, poly(L-histidine) was chosen as a proton sponge polymer. Herceptin, which specifically binds to HER2 antigen, was conjugated to the nanoparticles through click chemistry. The nanoparticles were characterized via dynamic light scattering (DLS) and transmission electron microscopy (TEM). Both methods showed the sizes of about 100 nm with a uniform size distribution. The pH sensitivity was assessed by drug releases and size changes at different pH conditions. As pH decreased from 7.4 to 5.2, the drug release rate accelerated and the size significantly increased. During in vitro tests against human breast cancer cell lines, MCF-7 and SK-BR-3 showed significantly increased uptake for Herceptin-conjugated nanoparticles, as compared to non-targeted nanoparticles. Herceptin-conjugated pH-sensitive nanoparticles showed the highest therapeutic effect, and thus validated the efficacy of a combined approach of pH sensitivity and active targeting. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Polymeric nanoparticles Breast cancer Active targeting pH sensitivity Herceptin1

1. Introduction The development of nano-scaled carriers as a drug delivery platform has made a tremendous difference in the fight against cancer (Park et al., 2002; Peer et al., 2007). Among these nanoscaled carriers, polymeric nanoparticles (NPs) made with biocompatible and biodegradable materials have been of particular interest (Xiong et al., 2009; Zhang et al., 2012; Zhu et al., 2014). One big advantage of NPs is that they can accumulate in the tumor tissue on systemic administration via the “enhanced permeability and retention” (EPR) phenomena (Maeda et al., 2000). The effectiveness of drug delivery system can be further enhanced by including targeting ligands to achieve active targeting, which will increase the specificity of a drug delivery system against a cancer of interest. There are many candidates of targeting ligands, such as folate (Yoo and Park, 2004), aptamers (Gu et al., 2008), and antibodies (Wartlick et al., 2004). Herceptin1

* Corresponding author. Tel.: +1 513 566 0018; fax: +1 413 556 0018. E-mail address: [email protected] (J.-Y. Lee). http://dx.doi.org/10.1016/j.ijpharm.2015.03.081 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

(Trastuzumab, Genentech) is a humanized antibody which specifically binds to Human Epidermal growth factor Receptor 2 (HER2/neu) and has been approved by FDA for the treatment of HER2-positive (HER2+) early-stage breast cancer and metastatic breast cancer (Hudis, 2007; Piccart-Gebhart et al., 2005; Smith et al., 2007). In addition, Herceptin and its fragments have also been conjugated to various drug carriers for active targeting against HER2 overexpressed breast cancer cells (Kim et al., 2011; Kirpotin et al., 2006; Zhao et al., 2012). Thus it has been chosen to provide our NPs anti-HER2 targeting. However, upon the internalization, the NPs are challenged against a new set of intracellular barriers. The NPs were internalized through endocytosis or pinocytosis. In either case, the NPs became localized inside the endosome and later would be trafficked into lysosome (Pack et al., 2005). Both of these two compartments are in acidic conditions, particularly the lysosome is further acidified to pH 4.5–5 and is full of degradative enzymes. If the internalized NPs cannot escape from these acidic compartments, they might eventually be digested by degradative enzymes or transported out of the cells via exocytosis. Then the cargos cannot be delivered to the cytoplasm or the nucleus where

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the drug is supposed to function. In order to address this issue, a “proton sponge” polymer, which promotes disrupture of endolysosome membrane, has been incorporated. Poly(L-hitidine) (PHis) is a perfect candidate for “proton sponge” polymer (Lee et al., 2003a,b,b; Pack et al., 2000; Putnam et al., 2003). The pKa of PHis is about 6.0, and it is made of an amino acid warranting biocompatibility (Patchornik et al., 1957). PHis also becomes hydrophilic when pH is below 6.0 as a result of protonation. This would induce the destabilization of the NP core, which makes NPs swell and triggers the fast release of encapsulated drugs. PHis can not only disrupt the endolysosomal membrane but also release the payload with accelerated kinetics upon endocytosis. The goal of this study is to show the efficacy of a drug delivery system with active targeting with Herceptin conjugation and endolysosomal escape and pH triggered drug release with PHis incorporation. For the development of such a system, three polymer components were used: first, poly(D,L lactide-co-glycolide) (PLGA), as a model controlled-release; second, poly-Lhistidine (PHis), as a pH sensitive polymer; and finally, polyethylene glycol (PEG) as a model hydrophilic polymer with antibiofouling properties. Doxorubicin is classified as an anthracycline antitumor antibiotic and considered one of the most potent Food and Drug Administration-approved chemotherapeutic drugs. Doxorubicin was encapsulated in nanoparticles as a model drug in this study (Carvalho et al., 2009). The NPs made of the biocompatible amphiphilic triblock copolymer were examined for in vitro drug release rate, cellular uptake, and cytotoxicity. 2. Materials and methods 2.1. Materials Na-Boc-Nim-2,4-dinitrophenyl-L-histidine (Boc-His(DNP)-OH), thionyl chloride, triethylamine (TEA), N,N-Diisopropylethylamine (DIEA), acetone, dimethylformamide (DMF), N-hydroxysuccinimide (NHS), tetrahydrofuran (THF), Dichloromethane (DCM), coumarin 6, tris[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl]amine (TBTA), Copper sulfate pentahydrate, Sodium ascorbate and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC
HCl) were purchased from Sigma–Aldrich (St. Louis, MO). Poly(D,L-lactide-co-glycolide) (50/50, MW 15,000) with terminal carboxylate groups was purchased from Akina (West Lafayette, IN). mPEG-NH2 (MW 5000) and NH2-PEG-N3 (MW 5000) were purchased from Laysan Bio Inc., (Arab, Al). NHS-PEG-alkyne (MW 2000) was purchased from Jenkem Technologies (Plano, TX). Doxorubicin hydrochloride (Dox
HCl) was purchased from LC Laboratories (Woburn, MA, USA). Dialysis tube (Spectra/Por1 1, MWCO 6000-8,000) was purchased from spectrum (Irving, TX). Amicon1 Ultra-4Centrifugal filter was purchased from Millipore (Billerica, MA). Herceptin1 was received as a generous gift from Genentech (San Francisco, CA). Pall Nanosep1 (MWCO 300,000) and Acrodisc1 Syringe Filter (0.45 mm and 0.2 mm) were purchased from Pall Corporation (Port Washington, NY). 2.2. Synthesis of PLGA-PHis-PEG and PLGA-PEG block copolymers A tri-block copolymer, PLGA-b-PHis-b-PEG-azide, was synthesized by conjugating PLGA-COOH to H2N-PHis-b-PEG-N3. As shown in Fig. 1, Boc-His(DNP)-OH (4 g) was suspended in anhydrous 1,4dioxane (50 ml). Then thionyl chloride (4 ml) was dropwise added to the stirring solution. This reaction was performed under nitrogen atmosphere for 40 min. DNP
NCA HCl was precipitated out by adding ice-cold diethyl ester and collected by centrifugation (3000  g for 5 min). In order to purify DNP
NCA HCl, acetone was used to dissolve DNP
NCA HCl and un-dissolvable impurities were removed by filtration. Then DNP
NCA HCl was precipitated by

pouring acetone solution into ice-cold diethyl ester, and dried in vacuo (yield 86%). Then the purified DNP
NCA HCl was used for the polymerization. The ring-opening polymerization of DNP
NCA HCl was initiated by azide-PEG-amine. DNP
NCA HCl (1 g, 2.6 mmol) and sodium carbonate (0.41 g, 3.9 mmol) were added into 20 ml anhydrous DMF (sigma) and stirred for 1 h under nitrogen steam. Then azidePEG-amine (326 mg, 0.065 mmol) was added to the stirring mixture (Hu et al., 2013; Lee et al., 2003b; Liu et al., 2012). After the three-day reaction under nitrogen steam, the mixture was filtrated and the solvent was removed by rotatory evaporator. Then product was precipitated by pouring the residual into ice-cold diethyl ester and collected by centrifugation (3000  g for 5 min) and dried in vacuo (yield 43%). To conjugate H2N-Poly(Nim-DNP-histidine)-b-PEG-N3 to HOOC-PLGA, PLGA-COOH in DCM was activated by EDC
HCl and NHS. The reaction was stirred at room temperature for 60 min. The product was precipitated by pouring the solution into ice-cold methanol and then collected by centrifugation (3000  g for 5 min) and dried in vacuo (yield 86%). The coupling reaction was carried out in DMF in the presence of DIEA (Kamaly et al., 2013). The reaction was stirred for 12 h at room temperature. The co-polymer was then precipitated by pouring into ice-cold diethyl ether and collected by centrifugation. Then the deblocking reaction of DNP was conducted in anhydrous DMF (20 ml), in which PLGA-b-poly (Nim-DNP-histidine)-b-PEG-azide (400 mg, 0.01 mmol) was dissolved, and then 2-mercaptoethanol (2 ml, 27 mmol) was dropwise added to the stirring solution. This mixture was stirred under nitrogen gas at room temperature for 12 h. Then the product was precipitated with ice-cold diethyl ester and dried in vacuo (yield 82%). A di-block copolymer, PLGA-b-PEG-N3, was synthesized by conjugating N3-PEG-NH2 to PLGA-COOH using the same coupling method. 2.3. NP preparation and characterization 2.3.1. NP preparation The nano-precipitation method was employed for preparing NPs (Quintanar-Guerrero et al., 1998; Fessi et al., 1989; Legrand et al., 2007). The di-block copolymer, PLGA-b-PEG-azide, or tri-block copolymer, PLGA-b-PHis-b-PEG-azide, and the drugs (doxorubicin) or the fluorescent molecules (coumarin 6) were co-dissolved in DMF to reach at polymer concentration 5 mg/ml. Doxorubicin was acquired by mixing doxorubicin hydrochloride with three molar excess of triethylamine in DMF overnight. Then this solution was dropwise added into DI water with stirring (water:DMF = 5:1 by volume). Finally this mixture was transferred to a dialysis tube (MWCO 6000-8000) and dialyzed against DI water for 24 h to remove the organic solvent. During this process, the external medium was replaced with fresh solution every 2 h in the first 6 h and then every 6 h in the last 18 h. The resulting suspended NPs were passed through 0.45 mm syringe filter to remove aggregates and free drugs. Then, NPs were concentrated by ultrafiltration (3000 g, 15 min, MWCO 50,000 Da) and were resuspended in PBS. To determine the amount of Dox loading in the NPs, the UV absorbance was measured at 485 nm and matched with a created calibration curve (Sanson et al., 2010). 2.3.2. Herceptin conjugation by click chemistry After the NP was formed, Herceptin as a targeting ligand, was conjugated to the surface NPs through click chemistry for active targeting (Hong et al., 2009). Herceptin was first modified with the NHS-PEG-alkyne linker (MW 2000) to introduce alkyne group to the antibody (Thorek et al., 2009). Then the unconjugated PEG linkers were removed by ultrafiltration (3000 g, 10 min, MWCO

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Fig.1. Schematic of Herceptin-conjugated PLGA-PHis-PEG synthesis route.

50,000). The modified antibody was then conjugated to the NP to create the active conjugate by combining 1 ml of 1 mg/ml nanoparticles (i.e. 1 mg) and 1 mg of modified antibody with 10 ml of 10 mM copper sulfate pentahydrate, 10 ml of 50 mM sodium ascorbate, and 10 ml of 10 mM tris[(1-benzyl-1H-1,2,3triazol-4-yl) methyl]amine (TBTA) was incubated at 4  C for 24 h in phosphate buffer (50 mM, pH 7.2). This reaction is usually referred to as the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction (Hong et al., 2009). In order to separate the unbound Herceptin, centrifugal filters (MWCO 300,000) were used after the click chemistry reaction step and NPs were further washed with PBS twice. The samples were resuspended with PBS. The filtrate was collected and the Bradford assay was used to measure the unbound Herceptin concentration to determine the amount of Herceptin conjugated to the NPs (Bradford, 1976; Liu et al., 2010). 2.3.3. Dynamic light scattering (DLS) Dynamic light scattering was employed to measure the size of NPs (0.2 mg/ml). To measure sizes of the pH sensitive NPs at

various pH conditions, the NPs were exposed to buffers with predetermined pH values (0.2 mg/ml) for 24 h before the measurement. 2.3.4. Transmission electron microscopy (TEM) The surface morphology of the NPs was investigated by the FEI CM20 transmission electron microscopy (TEM). 10 ml solution of 1 mg/ml freshly made NPs in PBS was dropped on carbon-coated copper grids. Then the excess amount of liquid was removed by filter paper. Next the grid was immersed in staining solution (0.75% w/v uranyl acetate) for 2 min, and finally dried in fume hood (Booth et al., 2011). 2.3.5. Confocal laser scanning microscopy (CLSM) The morphology of the pH sensitive NPs (PN) encapsulating coumarin 6 in an aqueous solution was investigated by CLSM (Zeiss LSM 510). 10 ml of a mount medium (SlowFade1 Gold Antifade Reagent, lifetechnologies, Grand Island, NY) was added onto a glass slide and then 10 ml of 0.1 mg/ml PN was dropped onto

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Fig. 2. 1H NMR characterization of tri-block copolymer of PLGA-b-Phis-b-PEG (A) (solvent: DMSO-d6) and di-block co-polymer PLGA-b-PEG (B) (solvent: CDCl3).

the mount medium. A cover slip was placed on top of the sample and an excess amount of the medium was removed by filter paper. 2.4. In vitro tests 2.4.1. In vitro drug release The suspended NPs loaded with Dox was transferred into a dialysis membrane (MWCO 6000–8000). Then the dialysis tube was immersed in 20 ml of PBS. For pH sensitivity studies, pH in the PBS was adjusted to a predetermined pH value by 0.1 N HCl. At a predetermined time between 8 and 72 h, then the PBS was collected and replaced. The amount of Dox released from the dialysis tube at each release time point was determined by the UV absorbance at 485 nm. 2.4.2. Cell culture SK-BR-3 breast cancer cells (ATCC, Manassas, VA) with HER2 overexpression and MCF-7 breast cancer cells (ATCC) with moderate HER2 expression (Lewis Phillips et al., 2008), were employed to examine the targeting ability of Herceptin and efficacy of the overall drug delivery system. The McCoy's 5A medium (ATCC) supplemented with 10% FBS and 1% penicillinstreptomycin was utilized as the cell culture medium for SK-BR-3 cells. The Eagle’s Minimum Essential Medium (EMEM from ATCC) supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 mg/ml insulin was utilized as the cell culture medium for MCF-7 cells. Cells were cultivated in a humidified environment at 37  C with 5% CO2. 2.4.3. In vitro cellular uptake In order to quantify the cellular uptake of NPs, a fluorescence dye (coumarin 6) was encapsulated within the NPs. After nanoparticle formation, nanoparticles were washed with amicon-4 filter (3000 g, 15 min, MWCO 50,000 Da) three times to remove unencapsulated coumarin 6. Then the fluorescence intensity of coumarin 6 loaded nanoparticles was measured on a microplate reader. The amount of encapsulated coumarin 6 was

determined based on a calibration curve constructed. Cells were cultured, collected, counted and then transferred to a 96-well plate with 5,000 cells per well, and incubated overnight before the tests. Then the cell culture medium was replaced with a culture medium containing NPs (0.125 mg/ml) and incubated for a predetermined time of 5, 30, and 120 min. The NPs were sterilized by passing through 0.2 mm syringe filter prior to all the cell tests. After incubation, suspended NPs were removed and cells were washed with PBS three times to remove unbound NPs. Then cell membranes were permeabilized by cell lysis solution (50 ml 0.5% (v/v) Triton X-100 in 0.2 N NaOH). The cell lysates were transferred to 96-well black plate for measurement. Then a microplate reader (Victor3, PerkinElmer, Santa Clara, CA) was used to measure the fluorescence intensity from coumarin 6 loaded NPs in the desired wells with an excitation wavelength at 485 nm and an emission wavelength at 530 nm. The cellular uptake was expressed by the following equation: cellular uptake ¼

Isample  Inegative control Ipositive control  Inegative control

(1)

The positive control was fluorescence intensity in the NP sample solution along with cell lysis solution. The negative control was fluorescence intensity in the blank well, containing cell lysis solution.

2.4.4. Fluorescent microscope Cells were cultured on cover slips in 12-well plate with 50,000 cells per well, and then cells were incubated with different formulations of NPs and Lysotracker (lifetechnologies, Grand Island, NY) for 2 h. After the incubation, cells were washed with PBS three times and fixed by 70% ethanol for 15 min. The nucleus were stained by 40 ,6-diamidino-2-phenylindole (DAPI). Finally, 10 ml of a mount medium (SlowFade1 Gold Antifade Reagent, lifetechnologies, Grand Island, NY) was added onto glass slide and the cover slip containing NPs treated cells was placed on the glass slide.

Z. Zhou et al. / International Journal of Pharmaceutics 487 (2015) 81–90 Table 1 Particle sizes measured by dynamic light scattering (DLS) for PLGA-PEG nanoparticles (NP), PLGA-PHis-PEG nanoparticles (PN), Herceptin conjugated PLGA-PEG nanoparticles (NPH), and Herceptin conjugated PLGA-PHis-PEG nanoparticles (PNH) at pH 7.4. Data represent mean  standard deviation, n = 3.

Diameter (nm)

NP

PN

NP-H

PN-H

102.9  7.5

99.7  1.8

134.9  18.7

130.3  9.2

2.4.5. In vitro cytotoxicity (MTS assay) Cells were planted to a 96-well plate with 5,000 cells per well, and then cells were treated with different formulations of NPs. The same Dox concentration in all NP formulation samples was ensured by determining the UV absorbance at 485 nm. The cytotoxicity of NPs was examined by the MTS assay (Promega, Madison, WI) according to the protocol provided by a vendor. The cell viability was obtained using the following equation: cellviability ¼

A470;sample  A470;negativecontrol A470;positivecontrol  A470;negativecontrol

(2)

85

The positive control was the absorbance at 470 nm obtained from 5000 cells without treatment. The negative control was the absorbance obtained from a blank well, containing the cell culture medium and MTS reagents. 3. Results and discussion 3.1. Development of tri-block copolymer PLGA-PHis-PEG-azide for Self-Assembly of NPs To make biomaterials that can self-assemble to a pH sensitive nanoparticle, we have developed a tri-block copolymer, consisting of PLGA, PEG, and PHis by the block end-grafting strategy. As shown in Fig. 1, first, a di-block copolymer, poly(Nim-DNPhistidine)-b-PEG, was synthesized by the ring-opening polymerization of DNP NCA HCl, using azide-PEG-amine as a marco-initiator. Then PLGA was conjugated to poly(Nim-DNPhistidine)-b-PEG to form a tri-block copolymer, PLGA-b-poly(NimDNP-histidine)-b-PEG, through the carbodiimide-mediated coupling reaction. Finally, DNP group was de-blocked by

Fig. 3. TEM images of PLGA-PEG nanoparticles (NP, A) and PLGA-PHis-PEG nanoparticles (PN, B).

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Table 2 Particle sizes measured by dynamic light scattering (DLS) of PLGA-PEG nanoparticles (NP) and PLGA-PHis-PEG nanoparticles (PN) at different pH conditions after 24 h incubation at room temperature. Data represent mean  standard deviation, n = 3. Diameter (nm)

pH 7.4

pH 6.4

pH 5.2

NP PN

109.0  1.6 105.1  1.5

104.6  2.0 337.2  65.2

105.4  1.8 870.3  28.7

2-mercaptoethanol, giving PLGA-b-PHis-b-PEG. The proton NMR characterization of PLGA-b-PEG and PLGA-b-PHis-b-PLGA are shown in Fig. 2. The presence of PHis on the tri-block copolymer was observed from the 1H NMR peaks between 6.5 and 8.5 ppm. By integrating the area under the curve, the number of repeating units of L-histidine was determined to be 30. 3.2. Characterization of NPs The NPs were prepared through the one-step nano-precipitation method. The polymer was first dissolved in a water miscible solvent, DMF, then precipitated in water to form NPs. The PLGA and PHis formed the core of NPs due to their hydrophobicity. Hydrophilic PEG formed the shell to stabilize the NPs, and the end group of PEG, azide, was used to conjugate with Herceptin. The conjugation of Herceptin to the NPs was completed via a click chemistry route. The anti-HER2 targeting agent, Herceptin, was

first modified with NHS-PEG-alkyne to introduce the alkyne group, which was then reacted with the azide group on the surface of the NPs to form a linkage between the NP and Herceptin (Hong et al., 2009; Thorek et al., 2009). DLS was used to measure the size of the NPs formed. As shown in Table 1, both PLGA-PEG NPs and PLGA-PHis-PEG NPs are approximately the same size, 100 nm. As shown in the TEM images of Fig. 3, the NPs exhibited relatively a uniform particle size distribution. However, the sizes of the NPs observed through the TEM images were smaller than the diameters measured by DLS. This could be attributed to the shrinkage of the NPs during the drying process when TEM samples were made. Similar observations were previously reported (Liu et al., 2007; Tang et al., 2006; Zhang et al., 2004). For Herceptin conjugated particles, the size increases by approximately 30 nm for both types of NPs. This can be attributed to the addition of Herceptin layer to the NPs. TEM was also used to observe the morphology and to examine the size of both types of the NPs. The amounts of conjugated Herceptin determined by the Bradford assay were 0.359 mg Herceptin per mg of NP and 0.291 mg Herceptin per mg of PN. The TEM images of both NPH and PNH shown in Fig. S1 also show unnoticeable differences in the size and morphology after Herceptin conjugation. 3.3. pH sensitivity of NPs To assess the pH sensitivity of nanoparticles, studies about pH-dependent size changes and drug release rates were

Fig. 4. Confocal laser scanning microscopy (CLSM) fluorescence images of coumarin 6 encapsulated pH insensitive PLGA-PEG nanoparticles (NP) and pH sensitive PLGA-PHisPEG nanoparticles (PN) at pH 5.2(left) and pH 7.4 (right) in 50 mM phosphate buffer at room temperature.

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Fig. 5. In vitro pH-dependent doxorubicin release profiles from PLGA-PHis-PEG nanoparticles (PN) and PLGA-PEG nanoparticles (NP) encapsulating doxorubicin at 37  C.

Fig. 6. In vitro cellular uptake efficiency of coumarin-6 loaded PLGA-PEG nanoparticles (NP), PLGA-PHis-PEG nanoparticles (PN), Herceptin conjugated PLGA-PEG nanoparticle (NPH), and Herceptin conjugated PLGA-PHis-PEG nanoparticles (PNH) on SK-BR-3 (A) and MCF-7 (B) cells after 5 min, 30 min and 120 min incubation at 0.1 mg/ ml nanoparticle concentration at 37  C. Data represent mean  standard deviation, n = 3. *p < 0.05, ***p < 0.0005.

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Fig. 7. Fluorescence microscopy images showing the internalization of fluorescent nanoparticles in SK-BR-3 cells (2 h incubation). Column 1:DAPI channels showing the blue fluorescence from 40 ,6-diamidino-2-phenylindole (DAPI) stained nuclei; Column 2: Coumarin-6 channels showing the green fluorescence from coumarin-6 loaded nanoparticles; Column 3: Lysotracker channels showing the red fluorescence from Lysotracker labeled acidic organelle; and Column 4: Merged channels DAPI, Coumarin6 and Lysotracker. Arrowheads indicate the co-localized NPHs within endolysosomes.

performed. PN and NP were suspended in PBS at different pH values and the sizes of the nanoparticles were measured using DLS as listed in Table 2. The size of PN increased remarkably from 100 to 850 nm as pH decreased from 7.4 to 5.2. However, the size of NP was kept almost the same. In order to visualize the swollen nanoparticles, coumarin 6, a fluorophore, was encapsulated inside the nanoparticles because of its high biocompatibility and high fluorescence activity (Hu et al., 2007; Sun et al., 2008). It was confirmed that the replacement of coumarin 6 with doxorubicin did not change the size and morphology of the nanoparticles from the TEM images of coumarin 6-loaded nanoparticles (Fig. S2). The amounts of coumarin 6 encapsulated in NP and PN were 0.034 and 0.044%(wt), respectively. The morphology of PN was directly observed at the acidic (pH 5.2) and neutral (pH 7.4) conditions using the confocal laser scanning microscopy (CLSM). As shown in Fig. 4, the sizes of green dots at pH 5.2 were significantly larger than those at pH 7.4, which is consistent with the DLS measurements. In the case of NP, no noticeable difference in the size of green fluorescence was observed between the acidic pH and neutral pH conditions. Furthermore, the fluorescence of coumarin 6 at pH 5.2 was notably brighter than at pH 7.4. This is because at the acidic pH, PN was swollen and coumarin 6 was no longer confined inside the core of PN, thus decreasing the effect of self quenching behavior induced by an increased local concentration of the fluorescent marker (Yin and Bae, 2009). Next the drug release rates were measured from doxorubicinloaded NP and PN under three pH conditions at 37  C. Fig. 5 shows the drug release profiles from the doxorubicin-loaded PN and NP. PN fast released 80 and 60% of doxorubicin at pH 5.2 and 6.4 in 8 h, respectively, while the rest of the samples (i.e., NP at all three pH values of 5.2, 6.4, and 7.4 and PN at pH 7.4) did not show any significant difference. As expected, NP did not exhibit pH-dependent release profiles, and PN at pH 7.4 showed a release profile similar to that of NP. Furthermore, the impact of Herceptin conjugation on the drug release rate was studied (Fig. S3). The conjugation of Herceptin reduced the drug release rate due to an addition of the protein layer on the surface of the nanoparticles, which is in agreement with the results previously reported (Liu et al., 2010). 3.4. In vitro cellular uptake After validating the pH sensitivity of the PLGA-PHis-PEG nanoparticles (PN), an uptake study was performed to examine

Fig. 8. In vitro cell viability results determined by MTS assay of SK-BR-3 (A) and MCF-7 (B) cells treated with free doxorubicin (Dox), PLGA-PEG nanoparticles (NP), PLGA-PHis-PEG nanoparticles (PN), Herceptin conjugated PLGA-PEG nanoparticle (NPH), and Herceptin conjugated PLGA-PHis-PEG nanoparticles (PNH) after 48 h incubation at 37  C. Each well had 5,000 cells. Data represent mean  standard deviation n = 4. *p < 0.05 vs. NP, **p < 0.005 vs. NP, ***p < 0.0005 vs. NP.

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the targeting ability of Herceptin conjugated nanoparticles. Two breast cancer cell lines were used, SK-BR-3 and MCF-7. In addition, three incubation times were used to obtain a temporal profile for the binding of Herceptin conjugated nanoparticles. As shown in Fig. 6, all the nanoparticles have approximately the same level of uptake after 5 min of incubation. On the other hand, after 30 min of incubation, remarkable uptake differences between Herceptin conjugated nanoparticles (NPH) and plain nanoparticles (NP) were observed in both cell lines. Both pH sensitive and pH insensitive nanoparticles with Herceptin conjugation as targeting ligands had much higher and faster uptake than their counterparts without Herceptin conjugation, validating the high efficiency of Herceptin for targeting HER2. In the case of SK-BR-3 cells, the cellular uptake efficiency doubled with Herceptin conjugation after 30 min incubation, whereas the cellular uptake efficiency increased 50% for MCF-7 cells. In addition, the cellular uptake efficiency for SK-BR-3 was much higher than MCF-7. This is because SK-BR3 has a higher level of HER2 expression than MCF-7 (Lewis Phillips et al., 2008). Further increasing the incubation time from 30 min to 2 h did not show any significant difference in uptake, indicating that the cells might have reached saturation. In order to ensure that the response is derived from the nanoparticles after cellular uptake, the release of coumarin 6 from NP and PN was examined. The release study of coumarin 6 in NP and PN was conducted in the same manner as that for the doxorubicin release study. After 24 h incubation, only 7.3 and 9.7%(wt) of coumarin 6 were released from NP and PN. Hence it was assumed that most of coumarin 6 was not released from the nanoparticles during the cellular uptake study up to 120 min. To identify the final fate of the internalized nanoparticles, a fluorescence imaging study was carried out using SK-BR-3 cells incubated with nanoparticles for 2 h. Three different dyes of DAPI, coumarin 6, and Lysotracker were used to label nuclei, nanoparticles, and acidic organelle, respectively, as shown in Fig. 7. In the case of Herceptin conjugated pH insensitive nanoparticles (NPH), a high level of co-localization of nanoparticles (green) and endolysosomes (red) was exhibited, indicating that the NPH was confined in acidic compartments. However, for Herceptin conjugated pH sensitive nanoparticles (PNH), the green color was evenly distributed through the cytoplasm and there was no accumulation of PNH at a confined location. The orange spots derived from localized nanoparticles (green) in endolysosomes (red) were noticeable for NPH, but not for PNH. The escape of PNH from endolysosomes suggests that proton sponge should induce endolysosome disruption. 3.5. In vitro cytotoxicity The efficacy of the nanoparticles in cell viability was examined using MCF-7 and SK-BR-3 cell lines. Cells were treated with doxorubicin (Dox) encapsulated NP, PN, NPH, PNH, and free doxorubicin (Dox). The drug loadings of NP, PN, NPH, and PNH were determined to be 3.1, 3.5, 2.6 and 2.5% (wt/wt). Cells without treatment were served as a control. After 48 h of incubation, the viability of the cells was assessed by the MTS assay. As shown in Fig. 8, at a high drug dosage of 1 mg/well, cells were over-killed and there was no distinguishable difference observed in term of different NP formulation of treatment. However, at a doxorubicin dosage of 0.5 mg/well, there are clear trends in both cell lines. PN was shown to be more effective than NP as was expected. This can be attributed to the proton sponge effect of PHis which leads to proton sponge triggered endosome escape and subsequent drug release into cytoplasm. Herceptin enabled the nanoparticles to achieve enhanced internalization for HER2 expressed cells, thus increasing the accumulation of the drug inside the cells. In both cell lines, Herceptin conjugated PLGA-PHis-PEG (PNH) showed the best

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cell killing ability. Blank carries were also examined to ensure that the carrier itself does not induce cytotoxicity. At a high nanoparticle concentration of 1 mg/ml, the cell viability of MCF-7 and SK-BR-3 was well above 90% (Fig. S4). 4. Conclusions In this study, an HER2 targeted pH-sensitive drug delivery system was developed to demonstrate its efficacy of delivering doxorubicin to breast cancer cells. By including the pH sensitive block, poly(L-histidine), doxorubicin-loaded nanoparticles showed a pH dependent drug release profile by means of size change. Herceptin conjugation also resulted in faster and higher cellular uptake and toxicity for MCF-7 and SK-BR-3 cells. This study indicates that a combined mechanism of active targeting and controlled release dependent on acidic pH prompted the drug release and proton sponge triggered endosomal escape can synergistically promote the high efficacy of the HER2 targeted pH-sensitive drug delivery system. Acknowledgments This study was supported by the Department of Biomedical, Chemical, and Environmental Engineering Interdisciplinary Seed Grant and the College of Medicine Dean’s Planning Grant of the University of Cincinnati. Herceptin1 was provided by Genentech, Inc. under the material transfer agreement number, OR-212587. The authors appreciate their support and helpful discussions with Professors Jing-Huei Lee Jaroslaw Meller, and Jagjit Yadav. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.03.081. References Booth, D.S., Avila-Sakar, A., Cheng, Y., 2011. Visualizing proteins and macromolecular complexes by negative stain em: from grid preparation to image acquisition. J. Vis. Exp. e3227. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Carvalho, C., Santos, R.X., Cardoso, S., Correia, S., Oliveira, P.J., Santos, M.S., Moreira, P. I., 2009. Doxorubicin: the good, the bad and the ugly effect. Curr. Med. Chem. 16, 3267–3285. Quintanar-Guerrero, D., Allémann, E., Fessi, H., Doelker, E., 1998. Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers. Drug Dev. Ind. Pharm. 24, 1113–1128. Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., Benita, S., 1989. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int. J. Pharm. 55, R1–R4. Gu, F., Zhang, L., Teply, B.A., Mann, N., Wang, A., Radovic-Moreno, A.F., Langer, R., Farokhzad, O.C., 2008. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc. Natl. Acad. Sci. 105, 2586–2591. Hong, V., Presolski, S.I., Ma, C., Finn, M.G., 2009. Analysis and optimization of copper-catalyzed azide–alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Ed. 48, 9879–9883. Hu, J., Miura, S., Na, K., Bae, Y.H., 2013. pH-responsive and charge shielded cationic micelle of poly(L-histidine)-block-short branched PEI for acidic cancer treatment. J. Control. Release 172, 69–76. Hu, Y., Xie, J., Tong, Y.W., Wang, C.-H., 2007. Effect of PEG conformation and particle size on the cellular uptake efficiency of nanoparticles with the HepG2 cells. J. Control. Release 118, 7–17. Hudis, C.A., 2007. Trastuzumab – mechanism of action and use in clinical practice. New Engl. J. Med. 357, 39–51. Kamaly, N., Fredman, G., Subramanian, M., Gadde, S., Pesic, A., Cheung, L., Fayad, Z.A., Langer, R., Tabas, I., Cameron Farokhzad, O., 2013. Development and in vivo efficacy of targeted polymeric inflammation-resolving nanoparticles. Proc. Natl. Acad. Sci. 110, 6506–6511.

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