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Alendronate conjugated nanoparticles for calcification targeting
Colloids and Surfaces B: Biointerfaces 142 (2016) 344–350
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Alendronate conjugated nanoparticles for calcification targeting Nanying Li a,b , Juqing Song a,b , Guanglin Zhu a,b , Xuetao Shi b,∗ , Yingjun Wang a,∗ a b
National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510641, China Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China
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Article history: Received 18 August 2015 Received in revised form 3 March 2016 Accepted 4 March 2016 Available online 7 March 2016 Keywords: Poly(lactic-co-glycolic acid) Nanoparticle Dopamine Calcification-targeting
a b s t r a c t In this article, the synthesis of a novel calcification-targeting nanoparticle (NP) is reported, which is realized through dopamine self-polymerization on the poly(lactic-co-glycolic acid) (PLGA) particle surface and subsequent alendronate conjugation. Cell viability and proliferation tests confirmed that such particle has low cytotoxicity and good biocompatibility. Experiments were designed to observe whether the synthesized NPs can pass through an obstructive hydrogel and directly bind themselves to hydroxyapatite (HA) NPs (mimicking calcified spots) and HA porous scaffolds (mimicking calcified tissues); and the result was positive, indicating ingenious targeting of NPs on calcifications. The calcification-targeting NPs are expected to be with promising applications on calcification-related disease diagnoses and therapies. © 2016 Published by Elsevier B.V.
1. Introduction As a kind of pathologic bone metastases, calcification can happen in normal tissues like vascular, nervous, mammary tissues or cartilage, which is not easy to be detected. In vivo calcification is a result of calcium deposition, which is similar to embryonic osteogenesis [1–3]. Calcification may include both osteogenic and chondrogenic differentiation of cells [3]. In calcified tissues, many bone matrix proteins and growth factors are expressed, such as bone morphogenetic protein-2 (BMP-2) [4], osteoprotegerin (OPG) [5] and osteopontin [6], indicating the calcification progress similar to bone formation and thus hydroxyapatite (HA) plays a role as the major ingredient of calcified tissues. General impression about calcification is that it indicates a benign lesion, which will not cause too serious consequences threatening life. But researches demonstrated that calcification is a distinguishing feature of cancers such as human breast cancer [7] and prostate cancer [8]. Under this consideration, diagnosis delay may lead to patients’ condition worsened and even endanger their lives. Traditional methods on calcification detection mainly include magnetic resonance imaging (MRI)[9] and in vivo micro-computed tomography (CT) via electron beam [10] or X-ray [11]. However, these methods usually come with obvious drawbacks like low resolution and the images may be illegible against diagnoses.
∗ Corresponding authors. E-mail addresses: [email protected] (X. Shi), [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.colsurfb.2016.03.015 0927-7765/© 2016 Published by Elsevier B.V.
In recent years, nuclear medicine and fluorescence labelling have been widely studied on in vivo imaging. For instance, Montet et al. [12] used nanoparticles (NPs) bound with RGD peptides to detect integrins on BT-20 tumor cells, while gold nanoparticles were employed in-cell protein detection by Cognet et al [13]. In these cases, nanoparticles with homing ability can be considered ideal tools to be labelled for disease diagnosis and even therapy. Generally, nanoparticles have been exploited as effective drug carriers for decades. As for cancer treatment, anticancer drugs can be more effectively transported to tumor tissues after being conjugated to NPs, in compare with oral administration or directly injection [14–16]. Accordingly, NPs as anticancer-drug carriers are able to promote drug distribution in carcinoma tissues and simultaneously reduce side effects in normal tissues. As a matter of fact, due to the lack of functional groups, many material surfaces cannot be modified directly by fluorescent molecules or drugs. Motivated by adhesive proteins in mussels, Lee et al. [17] presented polydopamine (PDA) as an excellent coating material which can apply to a large amount of materials including metals, inorganic materials and polymers. This method is quite facile to be realized through merely immersing substrates into an alkaline buffer solution containing dopamine for hours. After being modified by dopamine, various molecules can be easily immobilized or conjugated onto the modified surfaces of materials. According to a study of Jiang et al. [18], heparin was immobilized onto a hydrophobic polyethylene (PE) porous membrane through coupling with a reactive PDA layer, which modified the membrane surface in advance. Results showed platelet adhesion and anticoagulation abilities of the PE porous membrane
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Fig. 1. Schematic diagrams of alendronate grafting on the surface PLGA nanoparticles through dopamine polymerization (A) and bone targeting of ALN-PDA-NP (B).
Fig. 2. (A) Atomic force micrographs (scale bar: 500 nm); (B) Particle size distribution graph; (C) Zeta potential graph of the three different particles.
were enhanced, indicating heparin was conjugated well with the PDA layer. Via attaching initiator on PDA coating carbon nanotubes (CNTs), polydimethylamino-ethyl methacrylate (PDMAEMA) brushes were formed by atom transfer radical polymerization on the CNT surface [19]. Liu et al. [20] synthesized core-shell Fe3 O4 PDA NPs as drug carriers, and anticancer drug bortezomib (BTZ) was successfully bound to the particles, exhibiting great control release properties. Through surface modification with PDA, materials with
great biocompatibility and biodegradability, such as poly(lactideco-glycolide acid) (PLGA), obtain the ability to overcome the lack of reactive groups and can therefore carry functional molecules for better medical applications. In this work, we designed a novel calcification-targeting nanoparticle through coating the PLGA NP with PDA and subsequently combining it with alendronate, which has been formerly researched as a bone-targeting reagent for its impressive ability of HA orientation [21]. In vivo experiments had
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Fig. 3. Transmission electron micrographs of PLGA NPs, PDA-NPs and ALN-PDA-NPs successively (scale bar: 300 nm).
been carried out to prove alendronate with strong homing capacity to assess bulk calcium mineral through circulation, despite free calcium among the body [22]. In addition, studies showed alendronate capable to reach extraskeletal calcification in soft tissues like vascular [23] or breast [24] tissue and thus exert an effect. As for the mechanism, it was proved that the bite distance of alendronate’s deprotonated oxygens, O ... O , between the two phosphonates is 2.9–3.1 Å, and this separation is within the range found for the oxygens in HA, which can be the ideal distance for the chelation of Ca2+ in HA [25]. Hence, alendronate shows superior affinity towards HA rather than other calcium containing minerals. Besides, previous study showed alendronate could reduce the invasive potentials of the osteosarcoma cell, indicating alendronate itself possesses anti-cancer effect [26]. The synthesized material is expected to act as a novel kind of calcification detecting probe to diagnose calcification-related diseases, and its drug-carrier role granted by PDA coating and PLGA nanoparticles may lead to various potential therapy applications.
functionalization through conjugation to the PDA coating layers. Briefly, 100 mg alendronate was dissolved into 50 mL deionized water and 500 mg PDA coated NPs were put in under constant stirring. After 5 h, the NPs were centrifuged at 17,000g and washed with deionized water for 3 times. 2.4. Particle characterization Particles were suspended in 1 mM phosphate buffer (pH 7.4), and their sizes and zeta potentials were measured by Malvern Zetasizer Nano ZS90 (UK). NP morphology was observed by Bruker MultiMode atomic force microscopy (US). After being stained by phosphotungstic acid, the NPs were observed by JEM-2100F transmission electron microscopy (JP). 2.5. Cell culture
2. Materials and methods
NIH/3T3 cells were obtained from ATCC (US) and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS).
2.1. Materials
2.6. Cell viability and proliferation evaluation
PLGA(Molecular weight range: 10,000–18,000; 50:50 (Lacide:Glycolide)), Polyvinyl alcohol (PVA), HA nanoparticles (<200 nm), dopamine hydrochloride and tris(hydroxymethyl) aminomethane were purchased from Sigma-Aldrich. Alendronate was purchased from Wako (Japan).
NPs, treated or not, were divided into different groups to be incubated with cells for 1, 3 and 5 days. To measure cell viability, Cell Counting Kit-8 (Sigma-Aldrich) was used. Briefly, the cell-counting solution (10% v/v in the culture medium) was added to each sample. After 2 h of incubation, absorbance of the medium at 450 nm was measured against the reference absorbance of 690 nm by a plate reader (BioTek, US). Cell number was calculated from a standard curve of absorbance of known cell number. Cell proliferation data was acquired using Live/Dead assay kit (Invitrogen). 2 L 2 mM Ethidium homodimer-1 and 0.5 L 4 mM Calcein AM was added in 1 mL DPBS (Gibco), in which specimens were incubated for half an hour. After that, the dyes were removed and the specimens were viewed using a fluorescence microscope.
2.2. Synthesis of PLGA nanoparticles PVA was dissolved in 5 mL of distilled water at a 0.5 wt% concentration, to which 500 L of dichloromethane (DCM) containing 30 mg of PLGA was added. Then the resulted fluid was immediately dispersed using an ultrasonicator for three bursts and each lasted for 10 s (40% amplitude). The emulsion was added to 20 mL of deionized water and stirred overnight to evaporate the remaining DCM. The NPs were then washed and collected via centrifugation at 17, 000g for 20 min. The NPs can be simply labelled through adding fluorescent dye like rhodamine into the organic phase before emulsion. 2.3. Particle surface modification The synthesized NPs were immersed in 1 mL of Tris buffer (10 mM, pH 8.5) containing 2 mg of dopamine for 3 h at room temperature under constant stirring, and collected subsequently as polydopamine (PDA) coated PLGA particles by centrifugation at 17,000g for 10 min. Alendronate was used for particles’ surface
2.7. Bone mineral affinity study HA nanoparticles (70 mg) was dispersed in 0.1% agarose (SigmaAldrich) solution (heated in a microwave oven for dissolution). The HA entrapped agarose hydrogel slab (2 cm × 2 cm) was generated by cooling down the solution in a teflon mold. Then the slab was put into the saline (0.9% sodium chloride solution) or DMEM/FBS solution containing PLGA NPs, polydopamine coated PLGA NPs (PDA-NPs) or alendronate conjugated PDA-NPs (ALNPDA-NPs), respectively. After 2 h, the hydrogel slab was taken out and washed completely for particles collecting, and the binding
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Fig. 4. (A) Fluorescently labelled cells incubated separately with NPs, PDA-NPs, and ALN-PDA-NPs, including a blank control group (cells were merely incubated on a culture plate without microspheres treatment); (B) Cell viability analysis (scale bar: 100 m); (C) Cell number histogram as cell proliferation test result.
rates of nanoparticles to HA particles were measured. In detail, the gel was taken out and washed, the washing liquor was reserved and the gel was put into the agarase buffer [10 mM Bis Tris-HCl (pH 6.5), 1 mM Na2EDTA] at about 42 ◦ C. After the gel was completely hydrolyzed, all the solutions containing the particles were mixed and centrifuged for the particles collection. For PLGA NPs and PDA-NPs, via collecting unbound NPs and comparing their mass with the total, the binding rates are easy to calculate. In regard to the ALN-PDA-NPs, of which the majority bound themselves with the HA particles, the unbound NPs were gathered and dissolved in perchloric acid to transform alendronate into ionized state. Further determination was executed through alendronate’s chelation with ferric ions, as the referred article presented [27]. Comparing the amount of alendronate from the unbound NPs and the total, the binding rates can be measured. Besides, ALN-PDA-NPs respectively loaded with rhodamine and fluorescein isothiocyanate dexamethasone (FITC-DXM) were simultaneously added into the hydrogel-immersing solution and fluorescent images were taken to observe the binding condition. It is worth indicating that the fluorescent molecules were combined with the inner PLGA NPs rather than the coating layers Another experiment employed an agarose hydrogel slab (5 cm × 5 cm), in which a HA porous scaffold (porosity is more than 80%; porous size is 100–800 m) was wrapped to mimic softtissue-wrapped bone mineral. The hydrogel was put into the saline or DMEM/FBS solution containing NPs, PDA-NPs or ALN-PDA-NPs respectively. The affinity of NPs toward HA was measured using the same method described above. In addition, ALN-PDA-NPs labelled
by rhodamine were added into the hydrogel-immersing solution and observed via fluorescence microscope after binding to the HA scaffold. 2.8. Statistical analysis All quantitative data were statistically analysed via t-test. Pvalues less than 0.05 was considered statistically significant for all analyses. 3. Results 3.1. Nanoparticle synthesis and modification After being dissolved in DCM, PLGA was added in PVA solution and underwent ultrasonication to form white emulsion. PLGA NPs were obtained via overnight stir of a mixture of the emulsion and a certain amount of distilled water and subsequent centrifugation. PDA was coated on NP surfaces through incubating the particles with a pH 8.5 solution of dopamine. The NPs turned black due to PDA oxidation and then ALN were conjugated on the NPs by incubation (Fig. 1A). 3.2. Nanoparticle morphology Atomic force microscope (AFM) micrographs (Fig. 2A and B) showed that NPs, PDA-NPs and ALN-PDA-NPs were all with round shapes and their sizes are in a range not more than 600 nm. Zeta
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Fig. 5. (A) Fluorescence labelled ALN-PDA-NPs were added into DMEM/FBS solution immersing a hydrogel with HA particles inside; (B) Binding rate of different NPs to HA particles in saline and cell culture medium; (C) Fluorescence graph of binding ALN-PDA-NPs and HA particles (scale bar: 30 m). (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
potential of each type of NPs was demonstrated as shown in Fig. 2C, notably, after coating PDA and conjugating alendronate, the zeta potential tended to be more negative. The transmission electron microscopy (TEM) images showed a great part of the particles were with similar parameters around 300 nm (Fig. 3).
3.3. Cell viability and proliferation Cell viability was measured after 1, 3 and 5 days of culture, which was realized via detecting the activity of mitochondrial dehydrogenases. Cell viability bar graphs (Fig. 4B) exhibited cells incubated with NPs, PDA-NPs, and ALN-PDA-NPs separately were with very similar viability slightly above 90%. The proliferation of cells was also examined after 1, 3 and 5 days of culture in vitro, followed by stained with the “Live/Dead” kit. The fluorescence microscope images are shown in Fig. 4. To be mentioned, for each group 5 images were taken and cell numbers were calculated on this basis. The results indicated cell numbers of each group were very close, which meant NPs, PDA-NPs and ALNPDA-NPs hardly effected cell proliferation and thus showed low cytotoxicity.
3.4. Calcification-targeting ability test of alendronate-conjugated NPs PLGA NPs, PDA-NPs and ALN-PDA-NPs were separately added into saline or DMEM/FBS solution respectively, in which there existed a hydrogel with HA NPs entrapped inside. After 2 h, the binding rate of each kind of NPs to HA particles in saline and cell culture medium was measured as shown in Fig. 5B. The binding rate of ALN-PDA-NPs to HA was measured in saline at around 70% and in cell culture medium at about 60%, which was significantly higher than that of PLGA NPs and PDA-NPs at less than 10%. Special treatment was employed on ALN-PDA-NPs and HA particles to stain them with rhodamine (red) and FITC-DXM (green color) respectively for fluorescent labelling. From Fig. 5C, red fluorescence spots and green ones were distributed at the same positions as the spots observed in the optical images, proving the accurate binding of ALN-PDA-NPs labelled by different fluorescence onto HA particles. For the other experiment, PLGA NPs, PDA-NPs and ALN-PDANPs were put into saline or DMEM/FBS solution respectively, in which a HA scaffold containing agarose hydrogel was incubated. The binding rate of each kind of NPs to HA scaffold in saline and cell culture medium was measured after 2 h as exhibited in Fig. 6B. The binding rate of ALN-PDA-NPs to HA scaffold was measured in
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Fig. 6. (A) ALN-PDA-NPs labelled with rhodamine were added into DMEM/FBS solution, in which a hydrogel containing HA scaffold was immersed; (B) Binding rate of ALN-PDA-NPs to HA scaffold; (C) Fluorescence graph of the labelled ALN-PDA-NPs on the scaffold inside inner hydrogel (scale bar: 100 m).
saline at around 80% and in cell culture medium at about 70%, obviously surpassing those of other NPs with a binding rate as low as less than 10% in each condition. ALN-PDA-NPs were labelled with rhodamine, and fluorescent microscope showed that there were intense red fluorescence inside the scaffold, which meant considerable amounts of stained ALN-PDA-NPs passed through the outer hydrogel to seek for the HA scaffold and ultimately bound to it (Fig. 6C). What is more, there was no observed fluorescence outside the scaffold.
4. Discussion PLGA nanoparticles were synthesized using solvent evaporation method. To introduce their smooth surface catechol groups, dopamine self-polymerization was performed in Tris buffer with a pH of about 8.5. Under alkaline condition, the catechol groups were easily oxidized into quinoid structure and thus could reacted with −SH2, NH2 or NH functional groups according to Michael addition or Schiff-base reaction [28–30]. Alendronate as a bisphosphonate drug used for osteoporosis therapy and several other bone diseases can quickly bind to HA component after being absorbed by human body [31]. With a NH2 terminal group, alendronate can be readily conjugated to the PDA coating layers formed on PLGA NPs’ surfaces, which as a result granted these NPs special ability of bone affinity. This progress was simply achieved through incubating PDA-NPs in the solution of alendronate for hours. Via AFM and TEM images, it seemed the NPs surfaces turned a bit rough after PDA aggregation. As for the particle size measurement result discussed before, comparing with PLGA particles, there were more PDA-NPs and ALN-PDA-NPs over 300 nm than PDA-NPs in rate, which might ascribed to the thickness of the PDA layer increased along with dopamine polymerization. In addition, after
PDA coating, the particles’ Zeta potential tended to more negative, and such situation was more obvious via alendronate conjugation, which meant these series of modification did improve the dispersity of particles in suspension. The Zeta potential of ALN-PDPLGA-NPs is around −27 mV, which attributes to that the PDA is a kind of negative polyelectrolytes while ALN possesses negative charge in its ionized form in solution. Interestingly, it is found that as the HA component degrades, a bone crack releases ions with different diffusion coefficients and thus an electric field pointing outwards away from the crack is generated [32]. In consequence, the ALN-PDA-NPs with negative charge could be attracted by bone cracks and act as bone-crack-detecting probes as well. A study showed that PDA coated nano-fiber scaffold accommodated human endothelial cells well with low cytotoxicity [33]. Researches on alendronate also proved that it is not harmful to normal tissue cells [34,35]. In consistent, cell viability tests results showed barely no influence on cell viability and proliferation was made by alendronate conjugation, PDA-coating layers and PLGA NPs themselves, which meant the NPs, treated or not, were with low cytotoxicity and thus suitable for in vivo applications. That is, surface modification via PDA coating smoothly granted PLGA NPs with considerable reactivity for different molecules to graft onto, while their great biocompatibility and biodegradability were reserved. According to the in vitro experiments performed using hydrogels, ALN-PDA-NPs precisely spotted the internal HA particles and HA scaffold mimicking calcified spots and tissue. Our results (Figs. 5 and 6) indicated that ALN-PDA-NPs gathered in the solution to seek and get themselves fitted on HA. This ability was very powerful that even the HA particles and scaffolds were wrapped by hydrogels, ALN-PDA-NPs were able to pass all the obstacles and bind themselves to the bionic calcified spots and tissues, which was proved by fluorescence microscope observation that the inner HA
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particles and scaffolds were covered with intense red fluorescence. Considering the results, an infer can be made that the synthesized particles are with capability to go through muscles and other soft tissues like blood vessels, thus this material is injectable. As mentioned in the introduction part, the goal of this study was to form nanoparticles with the capability to locate bones or calcification of tissues. The ability to pass through soft tissue is a critical point for the particles to achieve such functionality. It was reported aqueous CdTe quantum dots embedded silica nanoparticles had been used as fluorescence probes and successfully labelled the MG63 osteosarcoma cells [36]. Through fluorescence labelling, the ALN-PDA-NPs can also be used as fluorescence probes to detect calcification, a symptom of cancers like mammary carcinoma. What is more, with PDA layers coating on the NP surfaces, other medical molecules can not only be loaded into PLGA nanoparticles via emulsification, but also be conjugated to the particles and exert the therapeutic action after the particles locate microcalcification points of tumors or bone cracks. 5. Conclusions In this study, we synthesized alendronate conjugated PLGA nanoparticles with PDA coating on their surfaces, which were proved with low cytotoxicity and superior ability of HA seeking and binding. The drug coupling on the NPs did not require chemical activation of the particles surfaces; it was facilely prepared through incubating NPs in dopamine solution under alkaline condition and subsequently immersing them in alendronate solution. These particles can be used as calcification detectors and drug carriers for osteal disease therapy with chemically active coating layers on their surfaces. Acknowledgements This study was financially supported by grants from the National Basic Research Program of China (2012CB619100), the National Natural Science Foundation of China (51232002, 51502095) and the 111 project (B13039). References [1] H.A. Fleisch, R.G.G. Russell, S. Bisaz, R.C. Mühlbauer, D.A. William, Eur. J. Clin. Invest. 1 (1970) 12. [2] M.J. Glimcher, Anat. Rec. 224 (1989) 139. [3] M. Abedin, Y. Tintut, L.L. Demer, Arterioscler. Thromb. Vasc. 24 (2004) 1161.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Alendronate conjugated nanoparticles for calcification targeting Nanying Li a,b , Juqing Song a,b , Guanglin Zhu a,b , Xuetao Shi b,∗ , Yingjun Wang a,∗ a b
National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510641, China Department of Biomedical Engineering, School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China
a r t i c l e
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Article history: Received 18 August 2015 Received in revised form 3 March 2016 Accepted 4 March 2016 Available online 7 March 2016 Keywords: Poly(lactic-co-glycolic acid) Nanoparticle Dopamine Calcification-targeting
a b s t r a c t In this article, the synthesis of a novel calcification-targeting nanoparticle (NP) is reported, which is realized through dopamine self-polymerization on the poly(lactic-co-glycolic acid) (PLGA) particle surface and subsequent alendronate conjugation. Cell viability and proliferation tests confirmed that such particle has low cytotoxicity and good biocompatibility. Experiments were designed to observe whether the synthesized NPs can pass through an obstructive hydrogel and directly bind themselves to hydroxyapatite (HA) NPs (mimicking calcified spots) and HA porous scaffolds (mimicking calcified tissues); and the result was positive, indicating ingenious targeting of NPs on calcifications. The calcification-targeting NPs are expected to be with promising applications on calcification-related disease diagnoses and therapies. © 2016 Published by Elsevier B.V.
1. Introduction As a kind of pathologic bone metastases, calcification can happen in normal tissues like vascular, nervous, mammary tissues or cartilage, which is not easy to be detected. In vivo calcification is a result of calcium deposition, which is similar to embryonic osteogenesis [1–3]. Calcification may include both osteogenic and chondrogenic differentiation of cells [3]. In calcified tissues, many bone matrix proteins and growth factors are expressed, such as bone morphogenetic protein-2 (BMP-2) [4], osteoprotegerin (OPG) [5] and osteopontin [6], indicating the calcification progress similar to bone formation and thus hydroxyapatite (HA) plays a role as the major ingredient of calcified tissues. General impression about calcification is that it indicates a benign lesion, which will not cause too serious consequences threatening life. But researches demonstrated that calcification is a distinguishing feature of cancers such as human breast cancer [7] and prostate cancer [8]. Under this consideration, diagnosis delay may lead to patients’ condition worsened and even endanger their lives. Traditional methods on calcification detection mainly include magnetic resonance imaging (MRI)[9] and in vivo micro-computed tomography (CT) via electron beam [10] or X-ray [11]. However, these methods usually come with obvious drawbacks like low resolution and the images may be illegible against diagnoses.
∗ Corresponding authors. E-mail addresses: [email protected] (X. Shi), [email protected] (Y. Wang). http://dx.doi.org/10.1016/j.colsurfb.2016.03.015 0927-7765/© 2016 Published by Elsevier B.V.
In recent years, nuclear medicine and fluorescence labelling have been widely studied on in vivo imaging. For instance, Montet et al. [12] used nanoparticles (NPs) bound with RGD peptides to detect integrins on BT-20 tumor cells, while gold nanoparticles were employed in-cell protein detection by Cognet et al [13]. In these cases, nanoparticles with homing ability can be considered ideal tools to be labelled for disease diagnosis and even therapy. Generally, nanoparticles have been exploited as effective drug carriers for decades. As for cancer treatment, anticancer drugs can be more effectively transported to tumor tissues after being conjugated to NPs, in compare with oral administration or directly injection [14–16]. Accordingly, NPs as anticancer-drug carriers are able to promote drug distribution in carcinoma tissues and simultaneously reduce side effects in normal tissues. As a matter of fact, due to the lack of functional groups, many material surfaces cannot be modified directly by fluorescent molecules or drugs. Motivated by adhesive proteins in mussels, Lee et al. [17] presented polydopamine (PDA) as an excellent coating material which can apply to a large amount of materials including metals, inorganic materials and polymers. This method is quite facile to be realized through merely immersing substrates into an alkaline buffer solution containing dopamine for hours. After being modified by dopamine, various molecules can be easily immobilized or conjugated onto the modified surfaces of materials. According to a study of Jiang et al. [18], heparin was immobilized onto a hydrophobic polyethylene (PE) porous membrane through coupling with a reactive PDA layer, which modified the membrane surface in advance. Results showed platelet adhesion and anticoagulation abilities of the PE porous membrane
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Fig. 1. Schematic diagrams of alendronate grafting on the surface PLGA nanoparticles through dopamine polymerization (A) and bone targeting of ALN-PDA-NP (B).
Fig. 2. (A) Atomic force micrographs (scale bar: 500 nm); (B) Particle size distribution graph; (C) Zeta potential graph of the three different particles.
were enhanced, indicating heparin was conjugated well with the PDA layer. Via attaching initiator on PDA coating carbon nanotubes (CNTs), polydimethylamino-ethyl methacrylate (PDMAEMA) brushes were formed by atom transfer radical polymerization on the CNT surface [19]. Liu et al. [20] synthesized core-shell Fe3 O4 PDA NPs as drug carriers, and anticancer drug bortezomib (BTZ) was successfully bound to the particles, exhibiting great control release properties. Through surface modification with PDA, materials with
great biocompatibility and biodegradability, such as poly(lactideco-glycolide acid) (PLGA), obtain the ability to overcome the lack of reactive groups and can therefore carry functional molecules for better medical applications. In this work, we designed a novel calcification-targeting nanoparticle through coating the PLGA NP with PDA and subsequently combining it with alendronate, which has been formerly researched as a bone-targeting reagent for its impressive ability of HA orientation [21]. In vivo experiments had
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Fig. 3. Transmission electron micrographs of PLGA NPs, PDA-NPs and ALN-PDA-NPs successively (scale bar: 300 nm).
been carried out to prove alendronate with strong homing capacity to assess bulk calcium mineral through circulation, despite free calcium among the body [22]. In addition, studies showed alendronate capable to reach extraskeletal calcification in soft tissues like vascular [23] or breast [24] tissue and thus exert an effect. As for the mechanism, it was proved that the bite distance of alendronate’s deprotonated oxygens, O ... O , between the two phosphonates is 2.9–3.1 Å, and this separation is within the range found for the oxygens in HA, which can be the ideal distance for the chelation of Ca2+ in HA [25]. Hence, alendronate shows superior affinity towards HA rather than other calcium containing minerals. Besides, previous study showed alendronate could reduce the invasive potentials of the osteosarcoma cell, indicating alendronate itself possesses anti-cancer effect [26]. The synthesized material is expected to act as a novel kind of calcification detecting probe to diagnose calcification-related diseases, and its drug-carrier role granted by PDA coating and PLGA nanoparticles may lead to various potential therapy applications.
functionalization through conjugation to the PDA coating layers. Briefly, 100 mg alendronate was dissolved into 50 mL deionized water and 500 mg PDA coated NPs were put in under constant stirring. After 5 h, the NPs were centrifuged at 17,000g and washed with deionized water for 3 times. 2.4. Particle characterization Particles were suspended in 1 mM phosphate buffer (pH 7.4), and their sizes and zeta potentials were measured by Malvern Zetasizer Nano ZS90 (UK). NP morphology was observed by Bruker MultiMode atomic force microscopy (US). After being stained by phosphotungstic acid, the NPs were observed by JEM-2100F transmission electron microscopy (JP). 2.5. Cell culture
2. Materials and methods
NIH/3T3 cells were obtained from ATCC (US) and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS).
2.1. Materials
2.6. Cell viability and proliferation evaluation
PLGA(Molecular weight range: 10,000–18,000; 50:50 (Lacide:Glycolide)), Polyvinyl alcohol (PVA), HA nanoparticles (<200 nm), dopamine hydrochloride and tris(hydroxymethyl) aminomethane were purchased from Sigma-Aldrich. Alendronate was purchased from Wako (Japan).
NPs, treated or not, were divided into different groups to be incubated with cells for 1, 3 and 5 days. To measure cell viability, Cell Counting Kit-8 (Sigma-Aldrich) was used. Briefly, the cell-counting solution (10% v/v in the culture medium) was added to each sample. After 2 h of incubation, absorbance of the medium at 450 nm was measured against the reference absorbance of 690 nm by a plate reader (BioTek, US). Cell number was calculated from a standard curve of absorbance of known cell number. Cell proliferation data was acquired using Live/Dead assay kit (Invitrogen). 2 L 2 mM Ethidium homodimer-1 and 0.5 L 4 mM Calcein AM was added in 1 mL DPBS (Gibco), in which specimens were incubated for half an hour. After that, the dyes were removed and the specimens were viewed using a fluorescence microscope.
2.2. Synthesis of PLGA nanoparticles PVA was dissolved in 5 mL of distilled water at a 0.5 wt% concentration, to which 500 L of dichloromethane (DCM) containing 30 mg of PLGA was added. Then the resulted fluid was immediately dispersed using an ultrasonicator for three bursts and each lasted for 10 s (40% amplitude). The emulsion was added to 20 mL of deionized water and stirred overnight to evaporate the remaining DCM. The NPs were then washed and collected via centrifugation at 17, 000g for 20 min. The NPs can be simply labelled through adding fluorescent dye like rhodamine into the organic phase before emulsion. 2.3. Particle surface modification The synthesized NPs were immersed in 1 mL of Tris buffer (10 mM, pH 8.5) containing 2 mg of dopamine for 3 h at room temperature under constant stirring, and collected subsequently as polydopamine (PDA) coated PLGA particles by centrifugation at 17,000g for 10 min. Alendronate was used for particles’ surface
2.7. Bone mineral affinity study HA nanoparticles (70 mg) was dispersed in 0.1% agarose (SigmaAldrich) solution (heated in a microwave oven for dissolution). The HA entrapped agarose hydrogel slab (2 cm × 2 cm) was generated by cooling down the solution in a teflon mold. Then the slab was put into the saline (0.9% sodium chloride solution) or DMEM/FBS solution containing PLGA NPs, polydopamine coated PLGA NPs (PDA-NPs) or alendronate conjugated PDA-NPs (ALNPDA-NPs), respectively. After 2 h, the hydrogel slab was taken out and washed completely for particles collecting, and the binding
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Fig. 4. (A) Fluorescently labelled cells incubated separately with NPs, PDA-NPs, and ALN-PDA-NPs, including a blank control group (cells were merely incubated on a culture plate without microspheres treatment); (B) Cell viability analysis (scale bar: 100 m); (C) Cell number histogram as cell proliferation test result.
rates of nanoparticles to HA particles were measured. In detail, the gel was taken out and washed, the washing liquor was reserved and the gel was put into the agarase buffer [10 mM Bis Tris-HCl (pH 6.5), 1 mM Na2EDTA] at about 42 ◦ C. After the gel was completely hydrolyzed, all the solutions containing the particles were mixed and centrifuged for the particles collection. For PLGA NPs and PDA-NPs, via collecting unbound NPs and comparing their mass with the total, the binding rates are easy to calculate. In regard to the ALN-PDA-NPs, of which the majority bound themselves with the HA particles, the unbound NPs were gathered and dissolved in perchloric acid to transform alendronate into ionized state. Further determination was executed through alendronate’s chelation with ferric ions, as the referred article presented [27]. Comparing the amount of alendronate from the unbound NPs and the total, the binding rates can be measured. Besides, ALN-PDA-NPs respectively loaded with rhodamine and fluorescein isothiocyanate dexamethasone (FITC-DXM) were simultaneously added into the hydrogel-immersing solution and fluorescent images were taken to observe the binding condition. It is worth indicating that the fluorescent molecules were combined with the inner PLGA NPs rather than the coating layers Another experiment employed an agarose hydrogel slab (5 cm × 5 cm), in which a HA porous scaffold (porosity is more than 80%; porous size is 100–800 m) was wrapped to mimic softtissue-wrapped bone mineral. The hydrogel was put into the saline or DMEM/FBS solution containing NPs, PDA-NPs or ALN-PDA-NPs respectively. The affinity of NPs toward HA was measured using the same method described above. In addition, ALN-PDA-NPs labelled
by rhodamine were added into the hydrogel-immersing solution and observed via fluorescence microscope after binding to the HA scaffold. 2.8. Statistical analysis All quantitative data were statistically analysed via t-test. Pvalues less than 0.05 was considered statistically significant for all analyses. 3. Results 3.1. Nanoparticle synthesis and modification After being dissolved in DCM, PLGA was added in PVA solution and underwent ultrasonication to form white emulsion. PLGA NPs were obtained via overnight stir of a mixture of the emulsion and a certain amount of distilled water and subsequent centrifugation. PDA was coated on NP surfaces through incubating the particles with a pH 8.5 solution of dopamine. The NPs turned black due to PDA oxidation and then ALN were conjugated on the NPs by incubation (Fig. 1A). 3.2. Nanoparticle morphology Atomic force microscope (AFM) micrographs (Fig. 2A and B) showed that NPs, PDA-NPs and ALN-PDA-NPs were all with round shapes and their sizes are in a range not more than 600 nm. Zeta
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Fig. 5. (A) Fluorescence labelled ALN-PDA-NPs were added into DMEM/FBS solution immersing a hydrogel with HA particles inside; (B) Binding rate of different NPs to HA particles in saline and cell culture medium; (C) Fluorescence graph of binding ALN-PDA-NPs and HA particles (scale bar: 30 m). (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
potential of each type of NPs was demonstrated as shown in Fig. 2C, notably, after coating PDA and conjugating alendronate, the zeta potential tended to be more negative. The transmission electron microscopy (TEM) images showed a great part of the particles were with similar parameters around 300 nm (Fig. 3).
3.3. Cell viability and proliferation Cell viability was measured after 1, 3 and 5 days of culture, which was realized via detecting the activity of mitochondrial dehydrogenases. Cell viability bar graphs (Fig. 4B) exhibited cells incubated with NPs, PDA-NPs, and ALN-PDA-NPs separately were with very similar viability slightly above 90%. The proliferation of cells was also examined after 1, 3 and 5 days of culture in vitro, followed by stained with the “Live/Dead” kit. The fluorescence microscope images are shown in Fig. 4. To be mentioned, for each group 5 images were taken and cell numbers were calculated on this basis. The results indicated cell numbers of each group were very close, which meant NPs, PDA-NPs and ALNPDA-NPs hardly effected cell proliferation and thus showed low cytotoxicity.
3.4. Calcification-targeting ability test of alendronate-conjugated NPs PLGA NPs, PDA-NPs and ALN-PDA-NPs were separately added into saline or DMEM/FBS solution respectively, in which there existed a hydrogel with HA NPs entrapped inside. After 2 h, the binding rate of each kind of NPs to HA particles in saline and cell culture medium was measured as shown in Fig. 5B. The binding rate of ALN-PDA-NPs to HA was measured in saline at around 70% and in cell culture medium at about 60%, which was significantly higher than that of PLGA NPs and PDA-NPs at less than 10%. Special treatment was employed on ALN-PDA-NPs and HA particles to stain them with rhodamine (red) and FITC-DXM (green color) respectively for fluorescent labelling. From Fig. 5C, red fluorescence spots and green ones were distributed at the same positions as the spots observed in the optical images, proving the accurate binding of ALN-PDA-NPs labelled by different fluorescence onto HA particles. For the other experiment, PLGA NPs, PDA-NPs and ALN-PDANPs were put into saline or DMEM/FBS solution respectively, in which a HA scaffold containing agarose hydrogel was incubated. The binding rate of each kind of NPs to HA scaffold in saline and cell culture medium was measured after 2 h as exhibited in Fig. 6B. The binding rate of ALN-PDA-NPs to HA scaffold was measured in
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Fig. 6. (A) ALN-PDA-NPs labelled with rhodamine were added into DMEM/FBS solution, in which a hydrogel containing HA scaffold was immersed; (B) Binding rate of ALN-PDA-NPs to HA scaffold; (C) Fluorescence graph of the labelled ALN-PDA-NPs on the scaffold inside inner hydrogel (scale bar: 100 m).
saline at around 80% and in cell culture medium at about 70%, obviously surpassing those of other NPs with a binding rate as low as less than 10% in each condition. ALN-PDA-NPs were labelled with rhodamine, and fluorescent microscope showed that there were intense red fluorescence inside the scaffold, which meant considerable amounts of stained ALN-PDA-NPs passed through the outer hydrogel to seek for the HA scaffold and ultimately bound to it (Fig. 6C). What is more, there was no observed fluorescence outside the scaffold.
4. Discussion PLGA nanoparticles were synthesized using solvent evaporation method. To introduce their smooth surface catechol groups, dopamine self-polymerization was performed in Tris buffer with a pH of about 8.5. Under alkaline condition, the catechol groups were easily oxidized into quinoid structure and thus could reacted with −SH2, NH2 or NH functional groups according to Michael addition or Schiff-base reaction [28–30]. Alendronate as a bisphosphonate drug used for osteoporosis therapy and several other bone diseases can quickly bind to HA component after being absorbed by human body [31]. With a NH2 terminal group, alendronate can be readily conjugated to the PDA coating layers formed on PLGA NPs’ surfaces, which as a result granted these NPs special ability of bone affinity. This progress was simply achieved through incubating PDA-NPs in the solution of alendronate for hours. Via AFM and TEM images, it seemed the NPs surfaces turned a bit rough after PDA aggregation. As for the particle size measurement result discussed before, comparing with PLGA particles, there were more PDA-NPs and ALN-PDA-NPs over 300 nm than PDA-NPs in rate, which might ascribed to the thickness of the PDA layer increased along with dopamine polymerization. In addition, after
PDA coating, the particles’ Zeta potential tended to more negative, and such situation was more obvious via alendronate conjugation, which meant these series of modification did improve the dispersity of particles in suspension. The Zeta potential of ALN-PDPLGA-NPs is around −27 mV, which attributes to that the PDA is a kind of negative polyelectrolytes while ALN possesses negative charge in its ionized form in solution. Interestingly, it is found that as the HA component degrades, a bone crack releases ions with different diffusion coefficients and thus an electric field pointing outwards away from the crack is generated [32]. In consequence, the ALN-PDA-NPs with negative charge could be attracted by bone cracks and act as bone-crack-detecting probes as well. A study showed that PDA coated nano-fiber scaffold accommodated human endothelial cells well with low cytotoxicity [33]. Researches on alendronate also proved that it is not harmful to normal tissue cells [34,35]. In consistent, cell viability tests results showed barely no influence on cell viability and proliferation was made by alendronate conjugation, PDA-coating layers and PLGA NPs themselves, which meant the NPs, treated or not, were with low cytotoxicity and thus suitable for in vivo applications. That is, surface modification via PDA coating smoothly granted PLGA NPs with considerable reactivity for different molecules to graft onto, while their great biocompatibility and biodegradability were reserved. According to the in vitro experiments performed using hydrogels, ALN-PDA-NPs precisely spotted the internal HA particles and HA scaffold mimicking calcified spots and tissue. Our results (Figs. 5 and 6) indicated that ALN-PDA-NPs gathered in the solution to seek and get themselves fitted on HA. This ability was very powerful that even the HA particles and scaffolds were wrapped by hydrogels, ALN-PDA-NPs were able to pass all the obstacles and bind themselves to the bionic calcified spots and tissues, which was proved by fluorescence microscope observation that the inner HA
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particles and scaffolds were covered with intense red fluorescence. Considering the results, an infer can be made that the synthesized particles are with capability to go through muscles and other soft tissues like blood vessels, thus this material is injectable. As mentioned in the introduction part, the goal of this study was to form nanoparticles with the capability to locate bones or calcification of tissues. The ability to pass through soft tissue is a critical point for the particles to achieve such functionality. It was reported aqueous CdTe quantum dots embedded silica nanoparticles had been used as fluorescence probes and successfully labelled the MG63 osteosarcoma cells [36]. Through fluorescence labelling, the ALN-PDA-NPs can also be used as fluorescence probes to detect calcification, a symptom of cancers like mammary carcinoma. What is more, with PDA layers coating on the NP surfaces, other medical molecules can not only be loaded into PLGA nanoparticles via emulsification, but also be conjugated to the particles and exert the therapeutic action after the particles locate microcalcification points of tumors or bone cracks. 5. Conclusions In this study, we synthesized alendronate conjugated PLGA nanoparticles with PDA coating on their surfaces, which were proved with low cytotoxicity and superior ability of HA seeking and binding. The drug coupling on the NPs did not require chemical activation of the particles surfaces; it was facilely prepared through incubating NPs in dopamine solution under alkaline condition and subsequently immersing them in alendronate solution. These particles can be used as calcification detectors and drug carriers for osteal disease therapy with chemically active coating layers on their surfaces. Acknowledgements This study was financially supported by grants from the National Basic Research Program of China (2012CB619100), the National Natural Science Foundation of China (51232002, 51502095) and the 111 project (B13039). References [1] H.A. Fleisch, R.G.G. Russell, S. Bisaz, R.C. Mühlbauer, D.A. William, Eur. J. Clin. Invest. 1 (1970) 12. [2] M.J. Glimcher, Anat. Rec. 224 (1989) 139. [3] M. Abedin, Y. Tintut, L.L. Demer, Arterioscler. Thromb. Vasc. 24 (2004) 1161.
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