WO2018042430A1 - Coatings of hydroxyapatite nanoparticles - Google Patents

Abstract

The invention provides a process for depositing a film of hydroxyapatite nanoparticles on at least a surface region of a substrate, by modifying neutrality of p H- sensitive residues present on the surface of the nanoparticles.

Description

COATINGS OF HYDROXY APATITE NANOPARTICLES

TECHNOLOGICAL FIELD

The invention generally relates to coatings containing hydroxyapatite.

BACKGROUND

Orthopedic and dental implants are vastly used in reconstructive medicine. Alloys, mainly titanium-based, are used to fabricate such implants. Coatings are often applied to improve the osteoconductivity and osteointegration. By far, coating of titanium with calcium phosphate (CaP) ceramics has become the dominant approach in this field. CaPs represent a family of materials consisting of various phases, among others, hydroxyapatite (HAp), a- and β-tricalcium phosphate (TCP), and octacalcium phosphate (OCP). The CaP coating increases both the bioactivity and biocompatibility of the implant, forming direct bonds with the adjacent tissue, thus enhancing osteointegration and promoting bone regeneration. Hydroxyapatite, HAp, Cas(P04)3(OH), has been studied extensively for a variety of applications, such as bone cements, bone fillers, bone tissue engineering, drug and gene delivery, etc.- this is due to its chemical and structural similarity to biological apatite, which is the prime inorganic constituent of bone.

Currently, HAp coatings can be formed by several processes, such as plasma spraying, laser pulse deposition, sol-gel coating, electrophoretic and electrochemical deposition [1]. Some of these techniques are expensive, form non-uniform coatings, and do not allow very precise control of the process. For example, plasma spraying, the most common method in industry today, is a non-line-of-sight technique which forms a variety of poorly crystalline phases on the implant's surface and introduces residual thermal stresses that eventually result in delamination and failure. Another example is electrophoretic deposition (EPD) of HAp [2,3]. This process involves applying high voltage to the substrate and therefore the HAp nanoparticles (NPs) must be calcined before deposition in order to evaporate adsorbed water [4]. Furthermore, EPD requires densification by sintering the coating at high temperatures [5] .

Electrochemical deposition is a low-cost, simple and flexible technique for coating conductive substrates [1,3,6]. Electrochemical deposition of HAp from aqueous solutions is associated with local altering of pH in the vicinity of the cathode surface as a result of water reduction, which causes the precipitation of CaP from solution [5,7,8]. BACKGROUND

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[4] I. Zhitomirsky, L. GalOr, /. Mater. Sci. Mater. Med. 1997, 8, 213.

[5] Z. C. Wang, F. Chen, L. M. Huang, C. J. Lin, /. Mater. Sci. 2005, 40, 4955.

[6] N. Eliaz, M. Eliyahu, /. Biomed. Mater. Res. Part A 2007, 80, 621.

[7] N. Eliaz, T. M. Sridhar, U. K. Mudali, B. Raj, Surf. Eng. 2005, 21, 238.

[8] N. Eliaz, W. Kopelovitch, L. Burstein, E. Kobayashi, T. Hanawa, /. Biomed.

Mater. Res. A 2009, 89, 270.

[9] D. Thiemig, A. Cantaragiu, S. Schachschal, A. Bund, A. Pich, G. Carac, C.

Gheorghies, Surf. Coat. Technol. 2009, 203, 1488.

[10] X. Pang, I. Zhitomirsky, Mater. Chem. Phys. 2005, 94, 245.

[II] S.-J. Park, J.-M. Jang, /. Nanosci. Nanotechnol. 2011, 11, 7167.

SUMMARY OF THE INVENTION

For the first time, titanium plates and implants were electrochemically coated by hydroxyapatite nanoparticles (HAp NPs). The proposed process involves oxidation of a protic solvent, i.e. water, to generate an acidic environment on the titanium surface. This causes discharge of the negatively charged HAp NPs and their irreversible deposition. Acids such as citrate and poly( acrylic acid) were used for dispersing the HAp NPs.

HAp coating was obtained both by potentiostatic and galvanostatic deposition, in which the thickness of the deposit was very well controlled by the applied potential or current and their duration. Coating of implants such as dental implant having complex geometries, as well as coating of implants with heat-sensitive materials that are contained or associated with the HAp NPs, without affecting their stability and efficacy, were successful, implying that the process developed is well adequate for industrial use. The coated surfaces, e.g., implants, exhibited high bioactivity, as demonstrated by the growth of an inorganic film upon soaking in SBF for 30 days at 37°C. The morphology of the soaked implant confirmed the formation of bone-like apatite layer, which resembles in vitro bone regeneration. This method has proven highly efficient, straightforward and economic, hence, could be well implemented in industrial use.

Hydroxyapatite, HAp, used in accordance with the invention is a calcium apatite (Caio(P04)6(OH)₂) of any known form, or a dicalcium phosphate of any known form, or a tri calcium phosphate of any known form, or an octacalcium phosphate of any know form, or calcium phosphates having a stoichiometry that ranges from CaO-2P₂05 to 4CaO-P₂05 and exhibiting solubility behavior, under acidic and basic conditions, similar to that of hydroxyapatite, or any other form known, that is presented for the purposes of the invention in the form of nanoparticles.

In a first aspect, there is provided a process for depositing a film of hydroxyapatite on at least a surface region of a substrate, the process comprising deposition of HAp nanoparticles, the surface of said nanoparticles being coated or associated with a plurality of pH-sensitive residues. The HAp may optionally be associated with at least one active or non-active agents that are associated to the surface of the nanoparticles, contained within the HAp matrix making the nanoparticles or contained within the nanoparticles. In some embodiments, the at least one active agent is a drug or a pharmaceutical, that is optionally heat-sensitive.

The HAp nanoparticles utilized in accordance with the invention may be any commercially available HAp (or generally any calcium phosphate) nanoparticles or such manufactured according to any one or more of the available processes for preparing same. In some embodiments, the nanoparticles are of any shape and size. In some embodiments, the nanoparticles have an averaged size, e.g., diameter, smaller than 200 nm. In some embodiments, the nanoparticles' averaged diameter is below 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40 30 or 20 nm. In some embodiments, the nanoparticles' diameter is between 20 and 200 nm.

In some embodiments, the nanoparticles are selected to be of a size stable in an aqueous medium.

In some embodiments, the HAp nanoparticles are pre-coated with a plurality of residues, or are modified to comprise surface regions that are associated with residues which charge or neutrality may be modified by adjustment of their pH. The residues may be pH-sensitive residues or pH-responsive residues that are protonatable by changing the acid strength (concentration) at their vicinity, or deprotonated under basic conditions. The pH-sensitive residues may be selected from carboxylates, alkoxides, amines, ammoniums, phosphates, phosphonates, phospites, sulfonates, sulfates, sulfinics, and others, each of which being in a charged form when presented on the surface of the nanoparticles.

In some embodiments, the residues are selected amongst any negatively charged groups that may be protonated in the presence of acids to afford neutral groups, e.g. such as a carboxylate or alkoxides, or positively charged groups having one or more acidic proton that is removable in the presence of a base, e.g., such as an ammonium group.

In some embodiments, the process comprising forming a dispersion of HAp nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of protonatable residues, e.g., pH-sensitive residues such as carboxylate or ammonium residues, and subsequently treating said dispersion under acidic conditions (in the case of negatively charged residues) or basic conditions (in case of positively residues) in the vicinity of the surface region of the substrate on top of which deposition is desired, to thereby cause deposition of said nanoparticles on the surface region of the substrate.

The pH-sensitive groups may be associated with the nanoparticles by reaction of the nanoparticles in the presence of an acid or a base form thereof, or in the presence of a base or an acid form thereof, under conditions causing surface association of the residues to the surface or surface region of the nanoparticles. In some embodiments, the pH-sensitive residue is derived from an acid that may be an organic acid or an inorganic acid. In some embodiments, the acid is a carboxylic acid. In some embodiments, the carboxylic acid is selected from methanoic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, pivalic acid, phenyl acetic acid, bromo acetic acid, chloro acetic acid, iodo acetic acid, 2-chloro butanoic acid, 4- methylpentanoic acid, citric acid, tartaric acid, ascorbic acid, benzoic acid, 4-nitro benzoic acid, carbonic acid, uric acid, taurine, acrylic acid and derivatives thereof, p- toluenesulfonic acid, triflic acid, aminomethylphosphonic acid, and others.

In some embodiments, the acid is citric acid and/or acrylic acid and/or polyacrylic acid.

When associated to the nanoparticles surface, the acid may be used in its base form, namely bearing a negative charge, namely unprotonated form to thereby form a film of pH-sensitive residues, which upon an increase in pH at their vicinity, undergo protonation to yield the acid forms. Similarly, basic conditions may be employed by utilizing bases such as amines alkoxides and others that have been protonated to be presented on the surface of the nanoparticles as positively charged residues, and thus pH sensitive. In some embodiments, the base is an organic base. In some embodiments, the organic base is an amine, for example selected from pyridine, alkyl amines, e.g., methyl amine, imidazoles and benzimidazoles, histidine, phosphazene bases and others.

In some embodiments, the pH-sensitive residues are a such materials having one or more acidic or basic functionality, as required. In some embodiments, the pH-sensitive residue is derived from a mono-acids; di-acids, tri-acids and polyacids. In some embodiments, the acid is a carboxylic acid and thus polyacids are such which comprise two, three, four...or more carboxylic acid groups.

In some embodiments, the pH-sensitive residues may be a combination of two or more such groups on the surface of the HAp nanoparticles.

In some embodiments, the base is an organic alkoxide.

The association of the pH-sensitive residues and the surface of the HAp nanoparticles may be achieved at any time prior to the deposition process. The association is typically physical and may be achieved by, e.g., sonication. In other cases the association may be chemical.

In some embodiments, the HAp nanoparticles are associated only with pH- sensitive residues; and therefore said to consist HAp or calcium phosphate and a plurality of pH-sensitive residues (materials) on their surface. In some embodiments, the nanoparticles are further associated with at least one active or non-active agent or material, as further defined herein. In such embodiments, the nanoparticles are said to consist HAp or calcium phosphate, a plurality of pH-sensitive residues (materials), as defined, and at least one active or non-active material.

As explained herein, with regard to some embodiments of the invention, the change in pH at the vicinity of the substrate to be coated with HAp is caused by applying a potential, which oxidizes water causing a reduction in the pH in vicinity of the implant surface. This results in protonation of pH-sensitive residues, e.g., carboxylic residues on the surface of the nanoparticles, thereby diminishing repulsion interactions among the nanoparticles and driving irreversible aggregation of the particles.

In some embodiments, the dispersion is an aqueous dispersion.

In some embodiments, the deposition is electrodeposition. In some embodiments, the deposition is electrochemical.

In some embodiments, the process comprises electrodeposition of HAp nanoparticles on a surface region of a substrate, the nanoparticles being coated or associated with a plurality of carboxylate residues, the process comprising contacting said substrate with a protic solvent dispersion (e.g., water or containing water) of said nanoparticles, under conditions permitting reduction in pH at the vicinity of the surface region of the substrate and protonation of the residues, to thereby cause deposition of the nanoparticles on the surface region of the substrate.

The invention further provides a process for forming a film of calcium phosphates (CP) nanoparticles on at least a surface region of a substrate, the process comprising treating a dispersion of CP nanoparticles coated or associated with a plurality of pH- sensitive residues, such as carboxylate or ammonium residues under conditions causing deposition of said nanoparticles on the surface region of the substrate.

The invention further contemplates a process for forming a film of calcium phosphates (CP) nanoparticles on at least a surface region of a substrate, the process comprising forming a dispersion of CP nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of pH-sensitive residues, such as carboxylate or ammonium residues, and subsequently treating said dispersion under acidic conditions or basic conditions (as explained herein and depending on the selection of pH-sensitive residues), to thereby cause deposition of said CP nanoparticles on the surface region.

In some embodiments, the deposition process comprises electrodeposition.

In some embodiments, the process comprising electrodeposition of CP nanoparticles on a surface region of a substrate, the nanoparticles being coated or associated with a plurality of carboxylate residues, the process comprising contacting said substrate with a water dispersion of said nanoparticles under conditions permitting reduction in pH at the vicinity of the substrate and protonation of the residues.

In some embodiments, the surface region is at least a surface region of a medical device or an implant.

The invention further provides a process for depositing calcium phosphates (CP) nanoparticles on at least a surface region of a substrate, the process comprising treating a dispersion of CP nanoparticles coated or associated with a plurality of pH-sensitive residues, such as carboxylate or ammonium residues, under conditions causing deposition of said nanoparticles on the surface region.

In some embodiments, the substrate is a metal substrate, optionally selected from a metal substrate which comprises titanium, stainless steel, cobalt, chromium, gold or alloys thereof. In some embodiments, the metal is titanium.

In some embodiments, the substrate is conductive. In some embodiments, the metal is selected to be conductive.

In some embodiments, the calcium phosphates (CP) is selected from hydroxy apatite (HAp), amorphous calcium phosphate and tricalcium phosphate of any known forms.

In some embodiments, the calcium phosphates (CP) is hydroxyapatite (HAp).

The invention further provides a film comprising CP, the film being formed by a process according to the invention.

Also provided are medical devices or implants comprising a film according to the invention. In some embodiments, the film is formed for promoting osteointegration and osteoconduction.

The invention further provides a process for promoting osteointegration or osteoconduction onto a surface of an implantable medical device, the process comprising forming a film of calcium phosphates (CP) nanoparticles on at least a surface region of a substrate by causing electrodeposition of said nanoparticles on a surface region of the medical device.

As used herein, the term "osteoconduction" refers to a process by which the coating formed on an implant permits or encourages new bone growth on its surface or in its pores, channels, or other internal voids. The implant of the invention is said to be " osteoinductive" when it can serve as a scaffold for new bone growth. The term further encompasses "osteoinduction" which refers to the process of stimulation of osteoprogenitor cells to differentiate into osteoblasts that then begin new bone formation. The term "osteointegration" refers to integration of osteoblasts (bone-forming cells) at a defect site of the host bone that is repaired utilizing an implant according to the invention as a framework upon which new bone is generated.

In some embodiments, the nanoparticles used in accordance with the invention are associated with or comprise at least one active agent. Unlike other processes used for coating a surface with HAp, the process of the invention permits inclusion of heat- sensitive materials that otherwise cannot be used in view of their thermal instability, and as other processes utilize high temperatures and less than mild application conditions. Thus, it is a purpose of the invention to provide a process for coating a substrate with HAp nanoparticles, the nanoparticles being associated with heat-sensitive active or non- active agents. In some embodiments, the heat-sensitive active agents are drugs or pharmaceuticals.

The heat-sensitive materials are generally materials that degrade at a temperature point to result in reduced activity (physiological effectiveness), that may be due to breaking of one or more covalent bonds in the materials and an associated change in its chemical characteristics. Degradation temperature may similarly result in other modifications in the chemical activity that may not necessarily be attributed to the breaking of a covalent bond. For example, degradation may occur at a temperature that contemplates e.g., the modification of a salt to a free acid or base and/or dehydration and/or desolvation.

A person versed in the art would know to identify materials that are thermally unstable or that may not be suitable for high temperature deposition processes, and would find the process of the invention better suited. The heat-sensitive materials, as defined, may be selected amongst biological materials that undergo degradation or decomposition at high temperatures (e.g., temperatures above body temperature, 37°C; or above room temperature, 23-29°C), materials that undergo phase change at higher temperatures and as a result can decompose or leach out from the HAp nanoparticles, or any other material which structure, form or function may be negatively affected when exposed to high temperatures. Such heat-sensitive materials include peptides, enzymes, amino acids, nucleic acids, small organic molecules, pharmaceutical drugs, such as those provided below, and others.

The association of the nanoparticles with the heat-sensitive materials, as any other material, active or non-active, may be achieved at any stage prior to the deposition process of the invention. The association may be as defined below.

In some embodiments, the active agent is at least one pharmaceutically active agent, e.g., a drug or a pharmaceutical. In some embodiments, the at least one pharmaceutically active agent is at least one drug selected from antibiotics, antibacterial agents, antifungal agents, antiviral agents, mitogenic growth factors, morphogenic growth factors, angiogenesis agents, anticancer agents, antiproliferative agents, anticlotting agents, antioxidants, analgesics, antiseptics, bioabsorbability/bioresorbability enhancers, bisphosphonates, calcitonins, chemotherapeutics, clotting agents, agents for treating pain, immune system boosters, immunosuppressants, immunomodulators, nutrients, statins, osteoclast inhibitors, antiinflammatory agents, osteogenic agents, agents promoting osteointegration or osteoconduction, and others.

A chemical or biological material, as an active agent, that is said to be osteoinductive, osteoconductive, osteointegrative, as defined herein, is such a material that can stimulate primitive, undifferentiated and pluripotent cells into the bone-forming cell lineage. Osteogenic agents are those that promote osteoblasts, as well as, osteoprogenitor cells, stem cells, and other cell types to be differentiated into mature osteoblasts, contribute to new bone growth at the bone implant site.

In some embodiments, the at least one active agent is an antibiotic. In some embodiments, the antibiotic agent is selected from cefazolin, cephradine, cefaclor, cephapirin, ceftizoxime, cefoperazone, cefotetan, cefutoxime, cefotaxime, cefadroxil, ceftazidime, cephalexin, cephalothin, cefamandole, cefoxitin, cefonicid, ceforanide, ceftriaxone, cefadroxil, cephradine, cefuroxime, ampicillin, amoxicillin, cyclacillin, ampicillin, penicillin G, penicillin V potassium, piperacillin, oxacillin, bacampicillin, cloxacillin, ticarcillin, azlocillin, carbenicillin, methicillin, nafcillin, erythromycin, tetracycline, doxycycline, minocycline, aztreonam, chloramphenicol, ciprofloxacin (Cip) hydrochloride, clindamycin, metronidazole, gentamicin, gentamicin sulfate (Gs), lincomycin, tobramycin, vancomycin, polymyxin B sulfate, colistimethate, colistin, azithromycin, augmentin, sulfamethoxazole, trimethoprim, and derivatives thereof.

In some embodiments, the at least one antibiotic is ciprofloxacin (Cip) hydrochloride, gentamicin or gentamicin sulfate (Gs).

In some embodiments, the at least one agent is selected from an autograft materials, allograft materials, ceramic-based bone substitutes, and blends and mixtures thereof. In some embodiments, the at least one agent is selected from corticosteroids, oxy sterols, compounds that upregulate intracellular cAMP, and compounds that impact the HMG coA reductase pathway and blends and mixtures thereof.

In some embodiments, the at least one active agent is a corticosteroid selected from budesonide, fluticasone propionate, fluoromethalone, halcinonide, clobetasol propionate, and blends and mixtures thereof. In some embodiments, the at least one active is an osteogenic material. The osteogenic material can be obtained from autogenic or allogenic sources and includes, autograft, autogenic bone marrow aspirate, autogenic lipoaspirate, allogenic bone marrow aspirate, allogenic lipoaspirate, and blends and mixtures thereof.

In some embodiments, the at least one active agent is associated with the nanoparticle material matrix, e.g., is part of the matrix making up the nanoparticle.

In some embodiments, the at least one active agent is contained within the nanoparticles, such that the active is coated with the nanoparticle material, e.g., a core- shell structure.

In some embodiments, the at least one active agent is associated with a surface region of the nanoparticle surface.

In some embodiments, the at least one active is surface exposed. In some embodiments, the at least one active is fully contained within the nanoparticles.

An implant used or manufactured in accordance with the invention is not limited and can take any form or shape. The implant may be selected from screws, such as bone screws, pedicle screws; tacks; nails, such as intramedullary nails, soft-tissue anchoring nails; pins, such as bone pins, immobilizer pins; plates, such as bone plates, maxillofacial plates; rods; clamps; staples; springs; stents; sutures; xenograft, heterograft, or allograft portions of bone, and others.

In some embodiments, the process comprises forming a dispersion of nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of pH-sensitive residues, e.g., carboxylate or ammonium residues, and subsequently treating said dispersion under acidic conditions or basic conditions, to cause deposition of said nanoparticles on a surface region of the implant or medical device.

In some embodiments, the process comprising electrodeposition of calcium phosphates (CP) nanoparticles on a surface region of the medical device, the nanoparticles being coated or associated with a plurality of pH-sensitive residues, e.g., carboxylate residues, the process comprising contacting said substrate with a water dispersion of said nanoparticles under conditions permitting reduction in pH at the vicinity of the substrate and protonation of the residues.

In some embodiments, the calcium phosphates (CP) is selected from hydroxyapatite (HAp), amorphous calcium phosphate and tricalcium phosphate. In some embodiments, the calcium phosphates (CP) is hydroxyapatite (HAp). As provided herein, the invention thus provides a process for forming a film of calcium phosphate (CP) nanoparticles on at least a surface region of a substrate, the process comprising treating a dispersion of CP nanoparticles coated or associated with a plurality of pH-sensitive residues under conditions causing deposition of said nanoparticles on the surface region, wherein said nanoparticles optionally comprising at least one active or non-active material.

Also provided is a process for forming a film of calcium phosphate (CP) nanoparticles on at least a surface region of a substrate, the process comprising forming a dispersion of CP nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of pH-sensitive residues, and subsequently treating said dispersion under acidic conditions or basic conditions to neutralize said pH-sensitive residues, to thereby cause deposition of said CP nanoparticles on the surface region, wherein said nanoparticles optionally comprising at least one active or non-active material.

In some embodiments, the pH-sensitive residues are selected from carboxylates, amines, ammoniums, phosphates, phosphonates, phospites, sulfonates, sulfates and sulfinics, each of which being in a charged form.

In some embodiments, the pH-sensitive residues are selected amongst negatively charged groups protonatable in presence of acid and positively charged groups having acidic proton removable in presence of a base.

In some embodiments, the deposition process comprises electrodeposition.

In some embodiments, the acidic or basic conditions involve application of a potential causing change in pH at the vicinity of the substrate, neutralization of the pH- sensitive residues and deposition on the substrate.

In some embodiments, the process comprises:

a. obtaining a dispersion of CP nanoparticles, optionally associated with at least one active or non-active material;

b. placing said dispersion in the vicinity of a substrate to be coated with said nanoparticles; and

c. modifying pH at the vicinity of said substrate to cause deposition of said nanoparticles on the substrate.

In some embodiments, modifying pH comprises application of voltage. As demonstrated hereinbelow, a change in the pH affects the ability to achieve deposition of the nanoparticles on a surface region of the substrate. The ζ-potential (zeta potential) for a nanoparticle dispersion should be greater than ±30 mV (either more positive or more negative than 30 mV).

In some embodiments, the process comprising electrodeposition of CP nanoparticles on a surface region of a substrate, the nanoparticles being coated or associated with a plurality of carboxylate residues, the process comprising contacting said substrate with a water dispersion of said nanoparticles under conditions permitting reduction in pH in the vicinity of the substrate and protonation of the residues.

In some embodiments, the conditions permitting reduction in pH involve applying positive potential.

In some embodiments, the surface region is at least a surface region of a medical device.

In some embodiments, the substrate is a metallic substrate.

In some embodiments, the substrate is conductive.

In some embodiments, the metallic or conductive substrate comprises a metal selected from titanium, stainless steel, cobalt, chromium, gold and alloys thereof.

In some embodiments, said calcium phosphate (CP) is selected from hydroxyapatite (HAp), amorphous calcium phosphate and tricalcium phosphate.

In some embodiments, said calcium phosphate (CP) is hydroxyapatite (HAp).

In some embodiments, the CP nanoparticles are associated with at least one active agent.

In some embodiments, the active agent is heat-sensitive.

In some embodiments, the active agent is at least one drug or pharmaceutical.

In some embodiments, in a process of the invention for forming a film of nanoparticles on at least a surface region of a substrate, the nanoparticles comprising calcium phosphate (CP) and at least one heat-sensitive material and are further coated or associated with a plurality of pH-sensitive residues, the process comprising treating a dispersion of the nanoparticles in the vicinity of the substrate under conditions causing neutralization of the pH-sensitive residues and deposition of said nanoparticles on the surface region of the substrate.

The invention further provides a process for forming a film of nanoparticles on at least a surface region of a substrate, the nanoparticles comprising calcium phosphate (CP) and at least one heat-sensitive material and are further coated or associated with a plurality of pH-sensitive residues, the process comprising treating a dispersion of the nanoparticles in the vicinity of the substrate under conditions causing neutralization of the pH-sensitive residues and deposition of said nanoparticles on the surface region of the substrate.

In some embodiments, the at least one active agent is selected from antibiotics, antibacterial agents, antifungal agents, antiviral agents, mitogenic growth factors, morphogenic growth factors, angiogenesis agents, anticancer agents, antiproliferative agents, anticlotting agents, antioxidants, analgesics, antiseptics, bioabsorbability/bioresorbability enhancers, bisphosphonates, calcitonins, chemotherapeutics, clotting agents, agents for treating pain, immune system boosters, immunosuppressants, immunomodulators, nutrients, statins, osteoclast inhibitors, antiinflammatory agents, osteogenic agents, and agents promoting osteointegration or osteoconduction.

In some embodiments, the at least one active agent is an antibiotic.

In some embodiments, the antibiotic is selected from cefazolin, cephradine, cefaclor, cephapirin, ceftizoxime, cefoperazone, cefotetan, cefutoxime, cefotaxime, cefadroxil, ceftazidime, cephalexin, cephalothin, cefamandole, cefoxitin, cefonicid, ceforanide, ceftriaxone, cefadroxil, cephradine, cefuroxime, ampicillin, amoxicillin, cyclacillin, ampicillin, penicillin G, penicillin V potassium, piperacillin, oxacillin, bacampicillin, cloxacillin, ticarcillin, azlocillin, carbenicillin, methicillin, nafcillin, erythromycin, tetracycline, doxycycline, minocycline, aztreonam, chloramphenicol, ciprofloxacin (Cip) hydrochloride, clindamycin, metronidazole, gentamicin, gentamicin sulfate (Gs), lincomycin, tobramycin, vancomycin, polymyxin B sulfate, colistimethate, colistin, azithromycin, augmentin, sulfamethoxazole, trimethoprim, and derivatives thereof.

In some embodiments, the antibiotic is ciprofloxacin (Cip) hydrochloride, gentamicin or gentamicin sulfate (Gs).

In some embodiments, the at least one agent is selected from an autograft materials, allograft materials, ceramic-based bone substitutes, and blends and mixtures thereof.

In some embodiments, the at least one agent is selected from corticosteroids, oxy sterols, compounds that upregulate intracellular cAMP, and compounds that impact the HMG coA reductase pathway and blends and mixtures thereof. In some embodiments, the at least one active agent is a corticosteroid selected from budesonide, fluticasone propionate, fluoromethalone, halcinonide, clobetasol propionate, and blends and mixtures thereof.

In some embodiments, the process is carried at room temperature.

The invention further provides a film comprising CP, the film being formed by a process according to the invention.

In some embodiments, the film further comprising at least one active agent, being optionally heat-sensitive.

Also provided is a film of nanoparticles comprising CP and at least one heat- sensitive material, the film being on a surface region of a substrate.

In some embodiments, the film is formed by a process according to the invention.

Also provided are medical devices or implants comprising films of the invention.

The films of the invention may be used for promoting osteointegration and osteoconduction.

The invention further provides a process for promoting osteointegration or osteoconduction properties to a surface region of an implantable medical device, the process comprising forming a film of calcium phosphate (CP) nanoparticles on the surface by causing electrodeposition of said nanoparticles onto the surface.

In some embodiments, the process comprising forming a dispersion of nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of pH-sensitive residues, and subsequently treating said dispersion under acidic conditions or basic conditions, to cause neutralization of the pH-sensitive residues and deposition of said nanoparticles on the surface region of the medical device.

In some embodiments, the process comprising electrodeposition of calcium phosphate (CP) nanoparticles on the surface region of the medical device, the nanoparticles being coated or associated with a plurality of pH-sensitive residues, the process comprising contacting said substrate with a water dispersion of said nanoparticles under conditions permitting reduction in pH in the vicinity of the substrate and protonation of the residues.

In some embodiments, the calcium phosphate (CP) is selected from hydroxyapatite (HAp), amorphous calcium phosphate and tricalcium phosphate.

In some embodiments, said calcium phosphates (CP) is hydroxyapatite (HAp). BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 provides a SEM image of HAp NPs (0.01% w/w) in an aqueous solution after 10 min sonication.

Fig. 2 provides an EDS spectrum of HAp NPs powder.

Fig. 3 is an X-ray diffraction pattern of synthesized HAp NPs powder. Indexing was done with respect to HAp (ICSD-PDF2 file 01-084-1998).

Figs. 4A-C present: Fig. 4A) ζ-potential, and Fig. 4B) particle size distribution of HAp NPs dispersed by Cit (dots) and PAA (squares) as a function of pH. Fig. 4C) is an image of Cit-stabilized HAp nanoparticle suspensions as a function of pH. The pH was changed by adding HC1 (0.5 M) or NaOH (0.5 M).

Figs. 5A-C present: Fig. 5A and 5B) XHR-SEM images of HAp NPs stabilized with Cit (10 mM) and electrochemically deposited at 2 V for 25 min. Fig. 5C) Thickness of the HAp NPs coating as a function of the applied potential for both dispersions (t = 25 min). Dots: PAA-stabilized HAp NPs, squares: Cit-stabilized HAp NPs.

Fig. 6 is an EDS spectrum of HAp deposition in ImM Cit solution.

Figs. 7A-B present Fig. 7A) XRD pattern of HAp coating on titanium, Fig. 7B) The corresponding overnight GIXRD pattern with Rietveld refinements.

Figs. 8A-B present FTIR spectra of HAp samples deposited with Cit and PAA (Fig. 8A) low wavenumbers (Fig. 8B) high wavenumbers.

Figs. 9A-B present the effect of time (Fig. 9A) and current density (Fig. 9B) on the thickness of coatings produced by HAp NPs stabilized by either Cit (squares) and PAA (dots).

Figs. lOA-C present SEM images at different magnifications of a dental implant electrodeposited with HAp NPs stabilized by Cit at 2 V for 25 min.

Figs. 11A-C present SEM images at different magnifications of a commercial dental implant coated with HAp NPs after 30 days soaking in SBF at 37 °C.

Figs. 12A-H present TEM (Figs. 12A-C) and SEM (Figs. 12D-F) of Gs-HAp, Cip-HAp and HAp, respectively, XRD (Fig. 12G) of pristine and drug-loaded HAp NPs, Zeta potential (H. l), particles size distribution (H.2) and digital photo (H.3) of Gs-HAp NPs dispersion in different alcoholic solvents.

Figs. 13A-C show SAED-TEM patterns of HAp (Fig. 13A), Gs-HAp (Fig. 13B), and Cip-HAp (Fig. 13C).

Figs. 14A-E present SEM (Figs. 14A-C) images in different magnification of Gs- HAp, Cip-HAp and GS-HAp+Cip-HAp NPs coatings, respectively. The dashed lines emphasizes the co-deposition of Cip-HAp and Gs-HAp. XRD (Fig. 14D) of drug-loaded HAp NPs coatings, FTIR spectrum (Fig. 14E) of drug-loaded HAp NPs.

Figs. 15A-B show the release profile of Gs-HAp (solid) and Cip-HAp (dashed). (Fig. 15A) Total drug amount, (Fig. 15B) Cumulative release percentage.

Figs. 16A-D are SEM images of Gs HAp (Fig. 16A,B) and Cip-HAp (Figs. 16C,D) before and after 25 days immersion in PBS at 37+1 °C.

Figs. 17A-D are SEM images of dental implant coated with Gs-HAp NPs; Fig. 17A before and Figs. 17B-D after 4 weeks immersion in SBF at 37+1° C.

Figs. 18A-B are SEM images of Gs-HAp coated (Fig. 18A) and uncoated (Fig. 18B) titanium implant after 4 weeks immersion in SBF solution at 37+1 °C.

Figs. 19A-F present a summary of agar diffusion tests performed with Pseudomonas aeruginosa bacteria. Fig. 19A - Average and standard deviation of inhibition diameters of different diffusion tests. Figs. 17B-F - Images of agar diffusion test of pristine HAp, Cip-HAp, Gs-HAp, pure Cip and pure Gs, respectively.

DETAILED DESCRIPTION OF EMBODIMENTS

As demonstrated herein, combining electrochemical deposition and HAp NPs offers both the advantages of a simple and straightforward technique with the formation of a controllable coating made of a HAp phase per se. For example, Thiemig et al. electrochemically deposited HAp-ZnFe nanocomposite film [9]. Pang et al. reported on EPD of HAp promoted by chitosan electrodeposition to create HAp-chitosan nanocomposite on stainless steel substrate in mixed water-ethanol solution [10]. Park et al. reported on electrodeposition of HAp NPs onto TiC nanotube layer under high potential (28 V) in an electrolyte containing N¾F and (NH4)H2PC¼ [11].

Here, the inventors present a novel approach for electrochemical deposition of pure HAp NPs, for coating titanium substrates (Scheme 1). The electrodeposition was successfully performed, using HAp NPs dispersed in aqueous solution using water- soluble stabilizing agents, such as tri-sodium citrate (Cit) and sodium poly acrylate (PAA). Deposition produces high purity, single phase, HAp coating under both potentiostatic and galvanostatic conditions. The process is driven by applying positive potential (<3 V), which oxidizes water causing a reduction in the pH in vicinity of the implant surface. This results in the protonation of the carboxylic residues of the dispersants and diminishes the repulsion interactions among the NPs, thus driving irreversible aggregation of the particles.

Results and Discussion

Preparation and characterization of HAp NPs

HAp NPs were synthesized via precipitation reaction from a solution of calcium nitrate into which diammonium phosphate solution was dropwise added. Fig. 1 shows SEM images of the HAp NPs in aqueous solution. It can be seen that the NPs are elongated. Dynamic light scattering gave an average size of ca. 94+40 nm. Furthermore, EDS and XPS analysis of the powder (Table 1) was performed.

Element at. %

EDS PS

Oxygen 68.3+2.5 60.2

Calcium 19.1+1.5 17.9

Phosphorus 12.5+0.1 11.3

Carbon — 10.6

Table 1. EDS and XPS element analysis of the HAp NPs.

In addition, EDS spectrum and element mapping of the powder is provided in Fig. 2. EDS showed that the ratio of Ca/P is 1.56+0.04. This value is somewhat close to that of stoichiometric HAp (1.67). It should be noted that EDS analysis is not recommended for unambiguous distinction between different CaP phases. In XPS measurement carbon is present in the spectra, which is related to CO2 contamination. The results indicate a similar Ca/P ratio of 1.58, which is yet somewhat lower than that of stoichiometric HAp. To further clarify the phase content, XRD measurements were conducted. Fig. 3 shows the XRD pattern of the HAp NPs. All reflections were assigned to the HAp phase (ICSD- PDF2 file 01-084-1998). No other CaP phases were detected. The degree of crystallinity of the HAp NPs calculated from the XRD is ca. 70%.

Unlike previous studies that involved the electrochemical deposition of HAp from ionic-molecular species, the technology being subject of the present application starts with HAp NPs dispersed in solution, involving their controlled deposition. In this case, stabilizing the HAp NPs was achieved by carboxylic acids, which enables their electrochemical deposition by induced protonation of the acid. The approach is based on applying a positive potential in aqueous solutions, which causes the formation of protons upon oxidation of water and, thus, the decrease of the pH on the electrode surface. Hence, the ζ-potential and size of the NPs were examined as a function of pH to assure that a decrease of pH causes their aggregation and precipitation. It should be noticed that this method differs substantially from EPD as the latter requires high voltage and is based on the migration of particles under electrical field.

HAp NPs were stabilized by acids such as Cit and PAA, the pKA of which is 3.13, 4.76, 6.39, and 4.25, respectively. This implies that the HAp NP dispersions are expected to be stabilized due to the strong negative repulsion between the NPs at pH > PK_A, and at the same time will be destabilized at pH < pKA. The change in the dispersion stability can be studied by measuring the ζ-potential and particle size distribution as a function of pH (Fig. 4).

It is evident that the decrease of pH affects the stability of both dispersions, i.e., stabilized by Cit and PAA. The ζ-potential shows a sigmoidal behavior where the particles attain negative potential of ca. -50 mV at pH > 8, while at pH < 3 the total charge of the NPs diminishes. The inflection point is at ca. pH 4, which is in good agreement with the pK_A2 of Cit and PK_AI of PAA. Furthermore, the change of the ζ-potential with pH matches the change in particle size. The sharp decrease of the particles' size at pH < 3 is due to their dissolution. This is also supported by the images of the dispersions (Fig. 4C). A stable dispersion is obtained as pH > 5, whereas lowering the pH yields initially an opaque solution (due to increasing the NPs size). All these results indicate that by altering the pH, namely, decreasing it below pH < 4-5, we destabilize the HAp dispersion and cause their precipitation. Electrochemical deposition and coating characterization

The next step comprised the electrochemical deposition of the stabilized HAp NPs dispersion onto Ti surfaces. Series of tests were conducted for the purpose of studying the effect of various applied potentials (1.5-2.5 V Ag/AgCl [1 M]) and duration on the deposition. The application of a positive potential drives the oxidation of water on the anode and generation of protons:

2H₂0 0₂ + 4H⁺ + 4e^" (1)

Figs. 5A and 5B show a typical SEM image of the Cit-stabilized HAp NPs on Ti deposited at 2 V for 25 min. It can be seen that the deposition is uniform and resembles the shape of the initial NPs (Fig. 1). It is conceivable that electrochemical deposition is also particularly suitable for template deposition using micrometer scale structures such as polystyrene particles. This template deposition may allow the selective deposition on implant's surface as well as the creation of microporous films. Varying the deposition potential had no effect on the nature of the deposit. Yet, it can be seen that the thickness of HAp coating increases as the applied potential is raised up to ca. 2 V, where it levels off (Fig. 5C). Therefore, it was decided to apply a potential of 2 V for the following experiments. Fig. 5C shows that the thickness of the PAA-stabilized coating is somewhat thicker at lower positive potentials than that by the Cit-stabilized NPs. This can be attributed to the difference in the pKA of PAA (4.25) and Cit (3.13). This difference implies that it should be easier to protonate PAA than Cit.

EDS element analysis of the coating (Table 2, lOmM) revealed relatively large amounts of carbon, indicating that the coating was enriched with Cit. The Cit deposition is attributed to the formation of Ti-Cit complex due to oxidation of titanium to Ti^"*⁴. Ti- Cit complex is water-soluble and, therefore, large amounts of Ti-Cit in the coating might induce the dissolution of the film and impair the stability of the HAp deposit in aqueous solution. Ti-PAA complex was also reported as water soluble. Element at.%

1 mM 5 mM 10 mM

Oxygen 62.0+1.5 62.7+1.2 64.9+2.3

Calcium 20.4+1 12.6+0.7 14.7+1.2

Phosphorus 12.4+1.2 7.7+1 9.0+0.4

Carbon 4.1+0.8 9.5+0.6 10.0+0.5

Titanium 1.1+0.3 3.7+2 1.2+0.8

Table 2. EDS element analysis of coatings electrodeposited at 2 V for 25 min in different Cit-stabilized electrolytes.

Therefore, different Cit concentrations were examined as shown in Table 2. Clearly, as the concentration of the Cit decreased, its content in the coating decreased respectively. Fig. 6 shows the EDS spectrum and elemental mapping of the coated substrate in low Cit concentration, which definitely confirms that the deposit is composed of pure HAp. We found that the HAp NPs dispersion was not affected, as long as the Cit concentration was higher than 1 mM. Therefore, further experiments were conducted with either 1 mM Cit or 0.03% (w/w) PAA.

Lowering the concentration of the ionic stabilizer, Cit or PAA, required the addition of an electrolyte to decrease the resistance of the solution. Thus, KNO3 (10 mM) was added. Further inspection of the EDS analyses (Table 2) showed good agreement with the expected ratio of Ca/P. The oxygen-to-calcium atomic ratio of stoichiometric HAp is 2.6, therefore only 53% of oxygen is attributed to the HAp while additional 9% of the detected oxygen comes from the Cit molecules. The oxygen-to-carbon ratio in Cit is 2.3, which should give rise to 3.9% of carbon, and therefore, is in accordance with the content that was found (4.1%). Hence, it is evident that most the carbon found in the coating originates from the Cit.

Fig. 7 A shows the XRD pattern of electrochemically deposited HAp coating on titanium. The four intense peaks are related to the titanium substrate (ICSF-PDF2 file 04- 002-2539). Fig. 7B shows grazing incidence XRD (GIXRD) with Rietveld refinements, which was performed overnight in order to observe the HAp signal while eliminating the intense peaks of the substrate. These results prove that the coating is composed of HAp. The spectrum of the HAp deposit reveals peak broadening, which is typical to nanocrystalline HAp. Indeed, the average crystallite size was between 12 and 18 nm. The hexagonal unit cell parameters were calculated and found to be a = 9.415 A and c = 6.870 A, which is in good agreement with the lattice parameters of stoichiometric HAp (a = 9 Alb A and c = 6.874 A, space group P6₃/m, based on ICSD-PDF2 file 01-084-1998). Figs. 8A and 8B show the furrier-transform infra-red (FTIR) spectra of the HAp coating stabilized with PAA and Cit. All low bands are attributed to HAp, as shown before. Moreover, a broad peak can be seen between 3100-3500 cm ¹ centered at 3260 cm^-1. This can be attributed to the stretching vibration mode of OH groups. Both are present in HAp, as well as in the PAA and Cit additives. No additional peaks were observed in the spectra.

Fig. 9A shows the effect of the duration of deposition on coating thickness upon applying a constant potential of 2 V for 25 min. It can be seen that the thickness of the HAp layer increases linearly, which indicates that the process is Faradaic and that the diffusion layer is not disrupted. This has been previously demonstrated by us in a potential-induced electrochemical deposition of sol-gel. Nevertheless, it was expected that the process will be self-limiting since the oxidation of water takes place at the Ti/electrolyte interface while deposition occurs at the HAp layer/electrolyte interface. Obviously, the distance between these two interfaces depends directly on the film thickness, which increases over time.

To assure a constant rate of deposition, it is preferable to perform the electrochemical deposition under constant current, as shown in Fig. 9B. Specifically, a constant current that varied between 0.1 and 0.5 mA cm^-2 was applied for 10 min. It is important to note that the current density during the potentiostatic deposition was ca. 0.15 mA cm^-2. It is evident that increasing the current density slightly increases the deposition rate for current densities below 0.4 mA cm^-2. At higher current densities, the rate of deposition increases more significantly; however, the Ti substrate was severely oxidized and the deposition was inhomogeneous.

The tensile stress to failure of the PAA-stabilized coating, as a standard measure of the adhesion of the coating to the substrate, was examined. The samples were first grid- blasted by alumina powder in order to increase the contact area between the glue and the samples, thus ensuring that the failure will not occur between the control sample and the glue. The stress to failure was 17.9+1.5 MPa. ESEM-EDS analysis was used to determine the locus of failure; both parts of the specimen were analyzed. In EDS analysis, Ca and P indicated the presence of HAp on the surface while Si and C were considered as indicators for the presence of glue on the surface. Ca and P were found on both the uncoated glued sample and the coated sample. Glue was found on both sides as well. These findings indicate that the failure was cohesive - either within the glue or within the coating. Hence, the adhesion of the coating to the substrate remained intact, and is expected to be higher than the value shown here. This result satisfies the requirement of the US FDA as well as of ASTM and ISO standards for an adhesion strength of at least 15 MPa.

Implant characterization and in vitro bioactivity test

To prove the applicability of our approach towards coating of medical devices, a commercial dental implant made of Ti-6A1-4V alloy was used. It was coated at 2 V for 25 min, as described above. Fig. 10 shows the SEM images of the coated implant. Clearly, the nanoparticulate coating covers well the surface of the implant; the complex geometry of the implant has no detrimental effect on the formation of the coating. Electroplating is very powerful for coating complex geometries, porous structures and non-line-of-sight surfaces. This benefit applies also to the HAp NP electroplating shown here. Since electron transfer is limited to a few nm from the electrode, the formation of a pH gradient will also follow the intimate structure of the surface. Hence, our method is highly appealing for industrial use.

The bioactivity of the coated implant plays a major role in determining the success or failure of the implantation. Therefore, the tendency of the coated implant to promote bone formation was tested by soaking the implant in a simulated body fluid (SBF) at 37 °C. Fig. 11 shows SEM images of the commercial dental implant coated with HAp NPs after soaking in SBF at 37 °C for 30 days.

It can be clearly observed that the morphology of the HAp NPs coating turned into porous interconnected network of HAp la^ er. 1 he elemental analysis of the coating by EDS is shown in Table 3. Normally, the formation of dense apatite layers on bioactive materials is observed after soaking in SBF for a certain I'me. However, the formation of interlinked apatite has rarely been reported. This porous apatite system can promote osseointegration and osteoconduction properties. Moreover, it is shown that after 30 days of incubation, processes of mineralization, nucleation and precipitation occurred presumably due to the interactions of the HAp NPs with the Ca⁺² and PO^^" ions in the SBF solution. Bone formation associated with HAp coating begins with surface dissolution of HAp, which releases Ca⁺² and PO^^" into the space around the implant and re -precipitating as new bone. The formation of apatite layer is based on the attraction of ions from the solution by positive (Ca⁺²) and negative sites ( PO^- and OH^~ ) on the HAp. This bioactive nature represents the sign of in vivo formation of bone bonding on the surface of the implant. The bioactivity test also reveals the stability of the electrochemical coating in SBF at 37 °C, which enables the biomineralization of bonelike apatite on a titanium implant. It can be observed that the presence of the organic stabilizer did not interfere with the formation of a new mineralized apatite layer. On the contrary, Cit is one of the bone components and has been reported to be a significant nucleation promoter of apatite crystals. Increased bioactivity of HAp sheets was observed after soaking in citrate -containing SBF compared to regular SBF. It can be assumed that the combination between HAp and Cit as two components of the bone (70 and 0.9 wt. , respectively) can lead to a superior bioactivity of the transplant. Similarly, PAA coating on titanium was reported as both enhancing anticorrosion and promoting osteoblasts function.

Before soaking After 30 days soaking

Element at.% Element at.%

Oxygen 62+4.7 Oxygen 58.0+5.6

Calcium 18.0+2.8 Calcium 23.0+5.2

Phosphate 11.4+4 Phosphate 15.0+2.4

Carbon 5.0+0.5 Titanium 1.4+0.5

Titanium 2.5+0.3 Aluminum 0.4+0.05

Aluminum 0.5+0.0 Magnesium 0.5+0. 2

Potassium 0.8+0.5

Sodium 0.7+0.1

Chloride 0.10+0.1

Table 3. EDS element analysis of HAp NPs coating deposited on commercial dental implant (made of Ti-6A1-4V) from Cit-stabilized dispersion after 30 days soaking in SBF at 37°C.

Elemental analysis (Table 3) shows elevated amount of calcium and phosphorus after soaking in SBF, which reflects the bioactivity of the HAp NPs coating. This is related to the enhanced deposition of Ca⁺² and PO^^" from the solution. The Ca/P ratio decreases from 1.6+0.1 before soaking to 1.5+0.2 after soaking, but this is not statistically different. The presence of CI^", K⁺ and Mg⁺² in the coating also indicates the biomineralization of the HAp coating by absorbing ions from the solution. The loss of carbon in the coating may be related to ion-exchange between Cit/PAA on the NPs surface to phosphate ions in the solution as part of the biomineralization of the implant.

Experimental Section

HAp NPs synthesis: HAp NPs were synthesized using a precipitation method. 4.722 g of Ca(N0₃)2 (ACS EMSURE®, Merck, Darmstadt, Germany) was dissolved in 18 mL of high purified water (Barnstead, Dubuque, Iowa, USA) using a magnetic stirrer. The pH of the solution was adjusted to 12 by adding 0.6 mL ammonium hydroxide (25%, Baker Analyzed^®, J.T Baker, Deventer, The Netherlands) and 17.4 mL water. 1.584 g of (NH₄)₂HP0₄ (BioUltra>99.0%, Sigma-Aldrich, St. Louis, Missouri, USA) was dissolved in 30 mL water while stirring. The pH of the solution was adjusted to 12 by adding 15 mL of concentrated ammonium hydroxide and another 19 mL deionized water. The diammonium phosphate solution was slowly added dropwise using a separatory funnel to the calcium nitrate solution while vigorously stirring. The slow addition resulted in a turbid suspension. The latter was boiled for 1 h. Then, the suspension was cooled to room temperature and left until the pH decreased to 7 (after ca. 3 days). The precipitated NPs were washed with water and centrifuged at 4000 rpm. This was repeated three times. The so obtained gel-like suspension was collected and froze-dried using liquid nitrogen. After 48 h, pure HAp powder was obtained. The HAp NPs were characterized by X-ray diffraction (XRD, Bruker, D9 Advance), X-ray photoelectron spectroscopy (XPS, Axis Ultra), and extreme high resolution scanning electron microscope (XHR-SEM, FEI Magellan™ 400L) equipped with energy-dispersive X-ray spectroscopy (EDS). All XRD results were compared to the ICSD (Inorganic Crystal Structure Data) files.

HAp suspension preparation: Suspension of HAp was prepared using either tri- sodium citrate (AnalaR^®, BDH Laboratory Supplies, Poole, England) or sodium polyacrylate (Mw ~ 5100 based on gel permeation chromatography, GPC, Aldrich) as dispersing agents. Specifically, 0.5% (w/w) HAp NPs were added to either 10 mM citrate (Cit) solution or 0.3% (w/w) polyacrylate (PAA) solution. Later on, HAp dispersions were prepared at low concentration of Cit or PAA (1 mM or 0.03% respectively) with the addition of 10 mM KN0₃ (ACS EMSURE^®, Merck, Darmstadt, Germany). Stable nanoparticle dispersions were obtained following sonication of 100 mL solution for 15 min at 100% amplitude, and pulse rate of 1 s on, 1 s off, using tip-sonicator (Sonics, Vibra cell). The suspension stability was examined by measuring the (zeta) ζ-potential and particle distribution size (Zetasizer, Malvern ZS).

Titanium surface pretreatment: Ti (Grade 4) plate and Ti-6A1-4V rod were purchased from Bramil LTD. The surface area of the Ti plate was 1.08 cm², and that of the Ti-6A1-4V rod was 1.27 cm². The Ti plates were manually ground on Grit 600 and Grit 4000 grinding paper (Microcut^®, Buehler, USA), rinsed in acetone, ethanol and water in ultrasonic bath (Elmasonic P, Elma) for 10 min, and etched in (40%)HF/(65%)HNO3 (2 vol % and 20 vol %, respectively) for 1.5 min. Commercial dental implants made of Ti-6A1-4V from SGS Dental Implants (Schaan, Liechtenstein) were tested. Electrochemical deposition: Electrochemical deposition was carried out using the potentiostat of a scanning electrochemical microscope (CH Instruments) in a conventional three-electrode cell. Platinum wire (35 mm long) was used as counter electrode, Ag/AgCl[l M] as reference electrode, pretreated Ti (Grade 4) or Ti-6A1-4V as working electrode. For each experiment, a fresh 17 mL dispersion (0.5% w/w HAp) was used. A constant potential, between 1.5-2.5 V vs. Ag/AgCl[l M] was applied for a given time. During deposition, the solution was moderately stirred. Then, the Ti surface was carefully withdrawn from the solution, washed with clean water and dried under atmospheric conditions. In another set of experiments, a constant potential of 2 V was applied for different times (5-25 min). Alternatively, a constant current density, between 0.1 and 0.5 mA cm^-2, was applied for a certain time. Commercial dental implants were electrochemically coated by applying 2 V for 25 min using PAA (0.03% w/w) as dispersing agent.

Coating characterization: The coated substrates were analyzed by XRD (2Θ = 15- 60° at step size 0.02 s at) and overnight glazing incidence XRD (GIXRD, 2Θ = 24-35°, step size 0.02 720 s at). High magnification images of the coated surface were taken both by XHR-SEM and by environmental SEM (ESEM, FEI Quanta 200FEG). The thickness of the samples was measured by profiler (KLA Tencor). Element analysis was performed by EDS. FTIR spectra were recorded using Bruker EQUINOX 55 in reflection mode. A liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector was used for high wavenumbers spectra, while deuterated triglycine sulfate (DTGS) detector was used for low wavenumbers spectra. The samples were scanned 2,500 times with 4 cm ¹ resolution. The spectra was recorded between 400 and 4500 cm^-1.

Adhesion test: The strength of adhesion of PP A- stabilized HAp coating to the metal substrate was tested by a standard tension test. Each test specimen was an assembly of a coated sample and a matching uncoated sample with exactly the same dimensions and surface pretreatment which included grit blasting by alumina powder (high purity white alumina powder from Calbex Mineral Trading, Inc., Henan, China). The blasting machine was model SandyPlus GD from Carlo DeGiordi (Italy). Blasting parameters were: grit size of F200-F180 (59-68 μπι), pressure of approximately 6 atm, and working distance of 3 cm or higher. The grit blast operation lasted until a dark grey shade evenly covered the sample. The sample was then washed in DI water and cleaned ultrasonically in acetone. The two parts of the assembly were bonded together by a thin layer of a 3M™ Scotch-Weld™ Epoxy Adhesive DP-420 Off-White, which was left to cure at room temperature for 24 h while exposing each assembly to a compression stress of 138 kPa (20 psi). The sample was held by grips of an MTS 20/M tensile machine. The tensile load was applied at a constant cross-head velocity of 0.5 mm min^-1. This velocity, which is slightly lower than that recommended, was found most suitable for the samples used in this study, where both the cross-section and the thickness of the coating were smaller than the values referred to in the art. Four assemblies were tensile tested. In addition to monitoring the maximum applied load, the locus of failure was determined by inspecting both parts of the assembly by means of ESEM-EDS.

Bioactivity Test: SBF (stimulated body fluid) was prepared according to a procedure that was elaborated elsewhere. The coated implant was soaked in SBF solution at 37°C for 30 days in thermostatic bath (Firstek B300). The morphology of the implant was examined by ESEM and XGR-SEM-EDS.

Drug-loaded HAp NPs synthesis

4.722 g of Ca(N0₃)₂ (ACS EMSURE®, Merck) was dissolved in 18 mL of deionized water (Barnstead, Dubuque) using a magnetic stirrer. The pH of the solution was adjusted to 12 by adding 0.6 mL ammonium hydroxide (25%, Baker Analyzed^®, J.T Baker) and 17.4 mL of water. For Gs-HAp NPs, 10 mg/mL of Gs (Bio-Reagent, Sigma- Aldrich) was added. For Cip-loaded HAp NPs, 10 mg/mL of Cip (>98%, BDL) was added. 6.6 mL of ammonium hydroxide was added in order to enhance the Cip dissolution. 1.584 g of (NH₄)₂HP0₄ (BioUltra>99.0%, Sigma) was dissolved in 30 mL water while stirring. The pH of the solution was adjusted to 12 by adding 15 mL of concentrated ammonium hydroxide and another 19 mL of deionized water. The Ca/P molar ratio in all the solutions was 1.667. The diammonium phosphate solution was slowly added dropwise using a separatory funnel to the calcium nitrate solution while vigorously stirring. The slow addition resulted in a turbid suspension. The latter was boiled for 1 h. Then, the suspension was cooled to room temperature and left until the pH decreased to 7 (after ca. 3 days) as a result of ammonia evaporation. Boiling for 1 h should not damage the antibacterial activity of the antibiotics due to their high thermal stability. The precipitated NPs were washed with water and centrifuged at 10,000 rpm for 5 min. The HAp NPs loaded with the antibiotics precipitate was collected and freeze-dried. The loaded HAp NPs were characterized by X-ray diffraction (XRD, Bruker, D9 Advance), X-ray photoelectron spectroscopy (XPS, Axis Ultra), high-resolution scanning electron microscopy (XHR-SEM, FEI Magellan™ 400L) equipped with energy-dispersive X-ray spectroscopy (EDS), and high resolution transmission scanning electron microscopy (HR-TEM, Tecnai F20 G2). All XRD results were compared to the ICSD (Inorganic Crystal Structure Data) files.

Drug-loaded HAp NPs suspension preparation

Gs-HAp NPs dispersion was prepared by adding 0.5% (w/w) NPs to various organic solvents: ethanol, 2-propanol, n-butanol (all BEAKER ANALYIZED® Reagent). Triethanolamine (TEOA, BEAKER ANALYIZED® Reagent) was added as a dispersing agent (4 mL/L) to the alcohols to increase the suspension stability. Cip-HAp NPs dispersion was prepared by adding 0.25% (w/w) NPs to 2-propanol. Combined dispersion of Gs-HAp and Cip-HAp was prepared by adding 0.25% w/w of each of the NPs to 2-propanol followed by the addition of TEOA (2 mL/L). Stable nanoparticle dispersions were obtained following sonication of 20 mL solution for 20 min at 90% amplitude, and pulse rate of 1 s on, 1 s off, using tip-sonicator (Sonics, Vibra cell). The suspension stability was examined by measuring the (zeta) ζ-potential and particle distribution size (Zetasizer, Malvern ZS).

Titanium surface pretreatment

Ti (Grade 4) plates and Ti-6A1^1V rods were purchased from Barmil Ltd. The surface area of the Ti plate was 1.08 cm², and that of the Ti-6A1-4V rod was 2.68 cm². The Ti plates were manually ground on Grit 600 grinding paper (Microcut^®, Buehler), rinsed in acetone, ethanol and water in ultrasonic bath (Elmasonic P, Elma) for 10 min, and etched in (30%)HF/(65%)HNO₃ (2 vol % and 20 vol %, respectively) for 1.5 min. Commercial dental implants made of Ti-6A1-4V from SGS Dental Implants (Schaan, Liechtenstein) were tested.

Drug loading determination

The amount of drug loaded into the HAp NPs was determined by a quantitative spectrophotometric method. Cip has maximum absorption at 271 nm. A calibration curve was prepared by measuring the absorbance at 271 nm of standard solutions of Cip (2-20 μg/mL) using a UV-Vis spectrophotometer (Evolution 201, Thermo Fisher Scientific). Gs has poor UV-Vis absorption, and therefore an indirect spectrophotometric method was required, using fluorescamine (>98%, Sigma) as a derivatizing agent. The maximum absorbance of the chromophoric product was determined at 391 nm. A calibration curve was formed by measuring the absorbance of series of Gs solutions (0-80 μg/mL) with a constant amount of fluorescamine. Detection limit (DL) was calculated based on the calibration graph according to ICH. Determination of the amount of either Gs or Cip in the HAp NPs was accomplished by dissolving 10 mg of the drug-loaded HAp NPs in 5 mL HC1 (0.5 M) followed by neutralization by 5 mL NaOH (0.5 M) and dilution with 40 mL of water. The absorbance of 5 mL of the sample was measured according to the maximum absorbance of the relevant drug, and the concentration was calculated based on the calibration curve. The loading percentage was evaluated by dividing the total drug amount in the solution by the weight of the drug-loaded NPs.

Electrophoretic deposition

Coating of the various samples was achieved by EPD (Major Science, Mini 300) in a conventional two-electrode cell. Pretreated Ti (Grade 4) or Ti-6A1-4V was used as anode, while stainless steel 316L with the same dimensions was the cathode. The two electrodes were placed parallel, the distance between them being approximately 5 mm. For each experiment, a fresh 20 mL dispersion (0.5% w/w Gs-HAp and 0.25% w/w Cip- HAp) was used. A constant potential of 80 V for Gs-HAp and 40 V for Cip-HAp was applied for 5 min. For co-deposition of Gs-HAp (0.25% w/w) and Cip-HAp (0.25% w/w), a potential of 60 V was applied for 5 min. After deposition, the samples were left to dry at ambient. Commercial dental implants were used also as substrates. The weight gain was determined by weighing the substrate before and after drying. The total amount of drug in the coating was calculated based on loading percent of the drug in the NPs. The coated Ti substrates were weighted before and after deposition using microanalytical balance (Mettler Toledo, XP26).

Coating characterization

The coated substrates were analyzed by XRD (2Θ = 10-60° at step size of 0.02 deg s^"1). High-magnification images of the coated surfaces were acquired by XHR-SEM and environmental SEM (ESEM, FEI Quanta 200FEG). The thickness of the samples was measured by a profilometer (PI 5, KLA-Tencor). Element analysis was performed by EDS and XPS. Fourier transform infrared (FTIR) spectra were recorded using Bruker Vertex 70V. Deuterated triglycine sulfate (DTGS) detector was used for the wavenumbers spectra. The samples were scanned 128 times at 4 cm ^_1 resolution. The spectra were recorded between 500 and 5000 cm^-1. The strength of adhesion Cip-HAp and Gs-HAp coatings to the metal substrate was tested by a standard tension test. Each test specimen was an assembly of a coated sample and a matching uncoated sample with exactly the same dimensions and surface pretreatment, which included grit blasting by alumina powder (high purity white alumina powder from Calbex Mineral Trading, Inc). The blasting machine was model SandyPlus GD from Carlo DeGiordi. Blasting parameters were: grit size of F200-F180 (59-68 μπι), pressure of approximately 6 atm, and working distance of 3 cm or higher. The grit blast operation lasted until a dark grey shade evenly covered the sample. The sample was then washed in DI water and cleaned ultrasonically in acetone. The two parts of the assembly were bonded together by a thin layer of a 3M™ Scotch-Weld™ Epoxy Adhesive DP-420 Off- White, which was left to cure at room temperature for 24 h while exposing each assembly to a compression stress of 138 kPa (20 psi). The sample was held by grips of an MTS 20/M tensile machine. The tensile load was applied at a constant cross-head velocity of 0.5 mm min^"1. This velocity was found most suitable for the samples used in this study, where both the cross-section and the thickness of the coating were smaller than the values referred to in the art. Three assemblies were tensile tested. In addition to monitoring the maximum applied load, the locus of failure was determined by inspecting both parts of the assembly by means of ESEM-EDS. Calculation of the drug wt % was conducted by microanalysis (Perkin- Elmer 2400 series II analyzer).

Drug release studies

Coated titanium substrates were immersed in phosphate-buffered saline (PBS, pH 7.4, Sigma) at 37+1 °C in humidity chamber (Memmert, HCP 108) for 25 days. Every 5 days the solution was replaced by a fresh PBS solution, where the former was spectrophotometrically analyzed. For each drug-loaded HAp coating, the experiment was conducted in triplicate. Bioactivity test

Simulated body fluid (SBF) was prepared according to a previous procedure. The coated implant was soaked in SBF solution at 37 °C for 4 weeks in a thermostatic bath (Firstek, B300). Every 5 days the SBF solution was replaced by a fresh one. The morphology of the implant was examined by ESEM and XHR-SEM-EDS.

Antibacterial activity

Gs-HAp and Cip-HAp coatings were scraped from the titanium substrates after EPD. The scraped coatings were introduced into PBS solution and sonicated for 1 h in order to release the antibiotics. Afterwards, the solutions were centrifuged for 10 min at 5000 RPM. The supernatants (20 μΕ) were dropped into agar plates, which contained Pseudomonas aeruginosa bacteria. The agar plates were incubated for 6 h at 37 °C. After incubation, inhibition zones were photographed and measured by a ruler. Each experiment was conducted three times. Pure Gs and Cip solution (100 μg mL^_1 in PBS) were used as positive control, and supernatants from scrapped pristine HAp coatings as negative control.

Results and Discussion

Preparation and characterization of drug-loaded HAp NPs

Drug-loaded HAp NPs were synthesized based on the precipitation reaction between calcium and phosphate ions under basic conditions where the antibiotics were introduced into the calcium solution prior to its dropwise addition to the phosphate solution. The interaction between calcium ions and Gs has been reported in various systems. At the same, it is unclear what the nature of this interaction is. Ca²⁺ ions are unlikely to electrostatically interact with Gs under the experimental conditions, i.e. pH~12, since the pK_A∞ = 10.18. Moreover, it has been reported that Gs does not chelate

Ca⁺² ions. On the other hand, Cip, which bears a carboxylic acid, is expected to interact with calcium ions by forming a stable complex. Therefore, the addition of Cip to calcium ions solution may enhance its loading into HAp NPs. The addition of the antibiotics to the Ca²⁺ solution was followed by the dropwise addition of phosphate, which resulted in the formation of a precipitate that was cleaned and characterized. Fig. 12 shows TEM images of Gs-HAp and Cip-HAp powders, which are made of NPs. It is evident that the addition of the antibiotics can affect the morphology of the NPs. Gs-HAp NPs exhibit elongated shape, which characterize crystalline HAp, while Cip-HAp NPs have more round morphology. The average size based on TEM was 20±8 nm and 40±12 nm for Gs-HAp and Cip-HAp, respectively. These results are somewhat different from dynamic light scattering (DLS) measurements of diluted dispersions, where the average size for Gs-HAp and Cip-HAp NPs was 150±40 and 370±35 nm, respectively. This might be related to the high tendency of small NPs to aggregate in the solution. The size of Cip-HAp aggregates is larger than Gs-HAp aggregates, which is in accordance with the nanoparticles size that form the aggregates.

It can be seen that Gs-HAp NPs are similar to pristine HAp NPs in terms of size and morphology, whereas Cip-HAp NPs have diverse structure. In addition, TEM images reveal that Cip-HAp NPs have a less crystalline structure compared to Gs-HAp and pristine HAp. As evidence, SAED patterns of the drug-loaded HAp NPs powder are shown in Fig. 13. The SAED pattern of pristine HAp shows a typical diffraction of polycrystalline materials, in which visible speckles are seen in the ring. On the other hand, it is hard to distinguish spots in the ring in SAED patterns of Gs-HAp and Cip-HAp. This implies that the crystal structure of both drug-loaded HAp NPs has lower crystallinity compared to pristine HAp. Comparison between the SAED of Gs-HAp and Cip-HAp shows that Gs-HAp has higher crystallinity. The inner ring of Gs-HAp is characterized by close spots, while in the outer ring, the spots are clearly seen. The SAED of Cip-HAp has more amorphous nature, where there are no visible spots in the ring. SEM images (Fig. 12) are in accordance with the TEM analysis.

XPS and EDS element analyses (Table 4) were conducted in order to determine the purity of the powder and to verify the encapsulation of the antibiotic into the NPs. The data for the pristine HAp NPs is also shown. The presence of elements such as fluorine and nitrogen can ensure the loading of the drugs. Both EDS and XPS confirm the presence of antibiotics in the HAp NPs. The incorporation of Gs in the HAp NPs is indicated by the presence of N and S, while the presence of F and N is attributed to Cip. A detailed discussion appears above.

Gs-HAp Cip-HAp

at % at %

EDS XPS EDS XPS EDS XPS

Ca 17.5 17.7±0.3 22.4 15.2±0.8 19.1 17.4±0.8

P 11.4 10.7±0.4 14.6 11.2±0.5 12.5 10.6±0.1

0 64.2 60.3±0.2 51.1 52.4±0.3 68.3 52.8±0.9

C 6.0 8.9±1 10.8 17.3±0.9 19.0±1.0

N BDT^* 1.3±0.2 BDT* 1.6±0.2

S 0.6

F 0.6 0.41±0.1

Table 4. EDS and XPS element analyses of Gs-HAp and Cip-HAp NP powders.

EDS shows that the atomic Ca/P ratio for both Gs-HAp and Cip-HAp is 1.53, which is somewhat lower than the 1.67 ratio that is expected for HAp. This might imply that the incorporation of the antibiotics into HAp impairs the purity of the phase. On the other hand, it should be noted that EDS analysis is not recommended for unambiguous distinction between different CaP phases. XPS analysis yields a Ca/P ratio of 1.65 and 1.35 for Gs-HAp and Cip-HAp NPs, respectively, which is closer to the expected ratio (1.64) Ca/P ratio for the HAp NPs. On the other hand, the low Ca/P ratio found for the Cip-HAp NPs may imply that the incorporation of Cip during the precipitation of HAp impairs its crystal structure, which is supported also by the SEM and TEM images. At the same time, we conclude that the incorporation of Gs into HAp NPs did not interfere with the formation of crystalline HAp, where the Ca/P ratio and structure are similar to the HAp NPs. Hence, we speculate that the structure of HAp NPs can be affected depending on the chemical structure of the antibiotic.

XRD measurements were conducted to further verify the phase of the powders and study the impact of the antibiotics addition on the crystallinity of the NPs. Fig. 12 shows the XRD patterns of Gs-HAp and Cip-HAp NPs powders. Comparison between synthesized HAp and Gs-HAp NPs shows that the incorporation of Gs to HAp does not significantly affect the crystal structure of the lattice. On the other hand, the addition of Cip to HAp NPs clearly results in defective crystal structure with substantially lower degree of crystallinity. The calculated degree of crystallinity was 70, 64, 31 % for pristine HAp, Gs-HAp and Cip-HAp, respectively. Thus, these results are in complete agreement with the above XPS, EDS, SEM and TEM findings. The difference in the crystallinity between Gs-HAp and Cip-HAp may be related to the mechanism by which the antibiotics interacts with calcium and phosphate ions. Gs is an aminoglycoside antibiotic rich in amine and hydroxyl moieties, and therefore it might interact with the -OH groups of HAp via hydrogen bonding or electrostatic interactions. We assume that the hydrogen bonding between the amines and hydroxyl moieties of Gs and the hydroxyl groups of HAp have a minute effect on the crystal structure of HAp. Indeed, it has been reported that the introduction of Gs during HAp precipitation had no effect on its crystalline structure. Cip belongs to the fluoroquinolones family, which is characterized by a fluorine atom bound to the central ring system, and a carboxylic residue. The most reasonable suggestion is that under basic conditions, the negatively charged carboxylic groups associate with the calcium ions. Due to the high concentration of the drug in the solution (10 mg/mL), the carboxylic group of the Cip is likely to interfere with the precipitation and formation of crystalline HAp. It is important to mention that the exact position of the drugs in the HAp NPs is not fully deduced.

The content of the drugs in the NPs was evaluated by spectrophotometric analysis, XPS and microanalysis (Table 5) in order to determine precisely the wt % of the drug in the NPs. The loading percentage based on XPS and microanalysis measurements was calculated based on the wt % of nitrogen as compared with the theoretical one. Clearly, all the analyses show good agreement. For Gs-HAp, there is a small deviation between the spectrophotometric calculation and both the XPS and microanalysis calculation. This may be a result of the chemical reaction, which is the basis of the spectrophotometric determination, and might increase the error.

Spectrophotometric Microanalysis XPS

(wt %) (wt %) (wt %)

Gs-HAp NPs 12.5 10.0 10.5

Cip-HAp N Ps 12.8 11.1 12.2

Table 5. Weight percentages of the antibiotics in the NPs.

It is very probable that the % of loading could have been increased. We have not attempted to optimize it. It depends on the initial concentration and obviously on the interactions between the drugs and the matrix. Yet, what is important is not only the % of loading by the % of release as will be discussed later.

Drug-loaded HAp dispersions

Low-molecular weight alcohols, such as methanol, ethanol, 2-propanol, and n- butanol, have been the most common solvents for the EPD of HAp NPs. TEOA has been reported as an efficient dispersant, therefore it had been added in order to increase the stability of the dispersions and the electrophoretic mobility of the particles. Firstly, we tried to obtain stable dispersions of the NPs in ethanol, 2-propanol, and n-butanol. The ζ-Potential and particle size distribution of Gs-HAp show that n-butanol is the optimal solvent for dispersing these NPs (Fig. 12). Clearly, the NPs possess positive charge in these solvents. The maximum concentration of Gs-HAp NPs, where a stable dispersion is still maintained, was 0.5% w/w. Based on similar experiments, 2-propanol was selected for dispersing Cip-HAp NPs without TEOA addition. The maximum concentration of Cip-HAp in the dispersion without further sedimentation was 0.25% w/w. EPD and coating characterization

After stabilizing the drug-loaded NPs in an organic solvent, we studied their EPD on Ti surfaces. Fig. 14 shows the SEM images (in different magnifications) of Gs-HAp and Cip-HAp NPs coatings. It can be seem that in both cases homogeneous coatings in terms of morphology and composition are obtained. Yet, there are some differences between the two layers. Specifically, the Cip-HAp NPs coating has less cracks and is less rough than Gs-HAp (Table 6). The difference is attributed to the thickness of the coating. We found that applying the same conditions (i.e. potential and time) resulted in substantially thicker films of Cip-HA NPs. Yet, the adhesion of the Cip-HAp NPs to the titanium was significantly worse than that of the Gs-NPs (Table 6). Therefore, we had to apply less negative potentials for the EPD of Cip-HAp, which yielded a 9.3 μπι- thick coating after 5 min, compared to 24.7 μπι-thick Gs-HAp NP coating after the same deposition time. Hence, we attribute the cracks formed in the Gs-HAp coating to its greater thickness. As the coating becomes thicker its tendency to form cracks is higher due to mechanical stresses related to shrinkage of the coating during drying.

Thickness (μιη) Roughness (μηη) Adhesion Deposit weight

(M Pa) (mg)

Gs-HAp 24.7±0.3 2.31 +0.26 5.8±0.7 7.7±0.6

Cip-HAp 9.3±1.3 0.37±0.07 13.7±2.1 2.1 +0.1

Table 6. Summary of selected properties of the two coatings.

These results indicate that the EPD is strongly affected by the drugs loaded into the HAp NPs. This is most likely due to the effect of the adsorbed drug on the ζ -potential, which clearly affects the rate of deposition. It is also probable that the lower crystallinity of Cip-HAp NPs influences the adhesion; however, we have no proof for this. Element Gs-HAp Cip-HAp

at % at %

Ca 17.3±0.1 13.4±0.1

P 9.8±0.2 9.0±0.1

0 56.0±0.5 45.2±0.1

C 14.6±0.6 27.1±0.3

N 1.2±0.5 1.5±0.3

F 0 1.4±0.2

Table 7. XPS analysis of Gs-HAp and Cip-HAp coatings on titanium.

XPS analysis of the coatings was conducted in order to ensure the presence of drug (Table 7). The detection of fluorine as well as nitrogen unambiguously indicates that each coating contains its relevant antibiotics. Comparison between the XPS analyses of drug-loaded HAp NPs before and after EPD (Table 4) shows that the element ratios in the coatings are similar, indicating that the drugs were not eluted during the EPD process. The total antibiotic amount in the coating was calculated by dissolving the coatings with HC1 (0.5 M) and determining the released drugs by spectrophotometry. The total amount of Gs and Cip in the coatings was 360±13 and 101±8 μg/cm², respectively. Recalling that the mass of the coating was 7.7 and 2.1 mg for Gs-HAp and Cip-HAp, respectively (Table 5), and taking into account the area that was coated (2.68 cm²), we get that the wt % of Gs and Cip is 12.5% and 12.9%, respectively. These values are similar to the wt % reported above (namely, 12.5% and 12.8%, respectively) for the NPs before coating. The total drug loading calculation via XPS reveals similar percentages compared to the NPs, i.e. 9.5% and 13.2% for Gs-HAp and Cip-HAp coatings, respectively.

Fig. 14 shows the XRD patterns of Gs-HAp and Cip-HAp coatings on titanium. The four intense peaks are related to the titanium substrate (ICSF-PDF2 file 04-002- 2539). While the pattern of Gs-HAp deposit shows the characteristic peaks of HAp, the HAp reflections in the Cip-HAp coating are barley seen. Poor crystallinity of Cip-HAp NPs and low thickness of the deposit are the main reasons of this. Moreover, calculation for HAp-Cip shows a small average crystallite size of ca. 63.1 A, while the average crystallite size of HAp-Gs was 129.2 A. The small crystallite size may also contribute to the broadened spectrum according to the Scherrer equation. FTIR spectrum (Fig. 14) shows the typical HAp peaks: PO4^3" vibration peaks at 565, 607, 962, 1026, 1078 and 1146 cm ¹. In addition, OH^" vibrations are observed at 630 and 3540 cm ¹. The spectrum of Cip-HAp exhibits different pattern. The peak at 1272 cm^{" 1} is attributed to the C-F vibration of Cip. The peak at 1627 cm^"1 is attributed to carbonyl C=0 stretching vibrations. The peak at 1584 and 1384 cm^"1 are assigned to stretching vibrations of COO^". The small peaks at 1546 and 1638 cm^"1 in the Gs-HAp spectrum may be associated with bending vibration of primary amines and the peaks between 2850- 3000 are related to CH2- stretching vibrations. In addition, in both spectra (Gs and Cip- HAp), the characteristics peaks of HAp are highly masked by the incorporation of the antibiotics by hydrogen bonding to P-OH groups. The spectrum of the mixed coating shows the incorporation of Cip. On the other hand, the determination of Gs in the coating is not conclusive.

One of the advantages of using pre-drug-loaded HAp NPs is the possibility to incorporate several types of antibiotics followed by their deposition in a single-step. The integration of a couple of drugs, such as Gs and Cip, can critically reduce the risk of implant infections due to the wider spectrum of antibacterial activity. In our research, we managed to disperse both Gs-HAp and Cip-HAp in the same solution and deposit them simultaneously to produce a drug-loaded coating with improved antimicrobial spectrum. Fig. 14 shows the drug-loaded coating composed of Gs-HAp and Cip-HAp. It can be seen that the coating is composed of both Gs-HAp and Cip-HAp NPs, as the former has elongated morphology whereas the latter is characterized by rounded morphology. XPS element analysis (Table 8) was conducted to assure the presence of both drugs. As mentioned before, the presence of F and N in the coating can verify that indeed the deposition of both drug- loaded HAp NPs was accomplished.

Element at %

Ca 13.5±1.4

P 8.3±0.8

0 53.0±1.0

C 22.5±2.5

N 1.8±0.5

F 0.5±0.1

Table 8. XPS element analysis of drug-loaded coating deposited from HA-Gs and HA-Cip dispersions.

Spectrophotometrically determination of the loading amounts of each drug in the coating has significant limitation due to similar functional groups, such as primary and secondary amines. Hence, the calculation of each drug was based on XPS analysis. Cip was determined by the F wt , whereas Gs by the N wt % after reducing the relative percent related to Cip. The calculated wt % of Cip and Gs in the mixed coating was ca. 8% and 4.4%, respectively.

Drug-release studies

Fig. 15 shows the kinetics of release of the antibiotics in vitro, i.e., the total drug amount released and the cumulative release percentage as a function of time per measurement. The data are average of triplicates. The Detection limit for Gs and Cip was 4.97 and 0.13 μg/mL, respectively. An initial burst in the first day can be seen in both profiles, which is followed by a slow release. For Cip-HAp coating, the release of the drug ceases after 10 day, as ca. 80% of the deposited drug is eluted to the aqueous solution. In contrast, Gs is continuously eluted from the coating for 25 days. However, only ca. 30% of the total loaded drug is released. This might be attributed to the thickness of the coating. The latter is smaller for Cip-HAp as compared with the Gs-HAp coating, which results in greater penetration of water into the coating and increase of drug dissolution. The thicker coating for Gs-HAp presumably affects also the total lower percentage of drug that is released. Yet, the remaining drug inside the coating may be eluted in vivo during the regeneration of ingrowing bone tissue towards the implant.

In order to assess the effect of the coating on the release, we examined also the release of the drugs from the antibiotics loaded HAp NPs dispersed in the same buffer as above. Specifically, 10 mg of Gs-HAp and Cip-HAp NPs were stirred in PBS at pH 7.4. After 2 hours, the solution was centrifuged, and 1 mL of the supernatant was analyzed spectrophotometrically. We found that approximately 94% of the drug was released during the first two hours. After 4 hours, the rest of the drug was eluted. This clearly shows that the difference between the release profile of Gs-HAp NPs and Cip-HAp NPs coatings is likely to be related to the morphology and structure of the deposit; thicker layers attain slower release of the encapsulated drug as compared with thin coatings.

SEM images (Fig. 16) of the drug-loaded HAp NPs coatings before and after 25 days immersion in PBS reveal interesting implications regarding the encapsulation of the drugs inside the NPs. It can be seen that the morphology of both Gs-HAp and Cip-HAp was changed. For both coatings, the size of the deposited NPs was shrank, especially for Cip-HAp, which the structure of the coating was significantly modified. The round structure of Cip-HAp was transformed into needle-like particles. This results may imply that indeed the drugs were encapsulated into the HAp NPs, and as a results of water penetration the drugs were dissolved into the aqueous media, which result in the particles shirking.

In-vitro bioactivity test

The tendency of a coated substrate to stimulate bone regeneration plays a significant role in the implantation success. HAp as the main component of the human bone promotes bone formation by adsorbing minerals from the body fluids and enhances the biomineralization of the coated implant. The bioactivity test was conducted on a commercial dental titanium implant. Fig. 17 shows SEM images of titanium implant coated with Gs-HAp before the immersion in SBF for 4 weeks. Clearly, the nanoparticulate coating deposited very homogeneously on the titanium implant, thus showing that complex geometries of substrate do not affect the morphology of the coating. Fig. 17 shows SEM images of the coated substrate after 4 weeks immersion in SBF at 37+1 °C. The bioactive nature of the coated titanium shows a significant adsorbing property. The precipitation of apatite crystals on the coated materials may resemble the biomineralization in vivo. It has been reported that the formation of an apatite layer in vitro by HAp suggests its ability to strongly interact with a living bone in vivo. Fig. 18 shows SEM images of Gs-HAp coated titanium implant and uncoated titanium implant after 4 weeks immersion in SBF solution. Clearly, biomineralization of the implant occurred only in the coated implant indicating the bioactive nature of drug-loaded coating. On the other hand, the morphology of the uncoated implant remained the same, without any indication of a precipitation of inorganic materials.

Further in-sight on the biomineralization of the Gs-HAp coating was gained from the EDS element analysis given in Table 4. It is evident that the layer that grew on the coated implant is primarily made of apatite, but contains also a low percent of other minerals. The presence of ions such as Na⁺, Mg⁺² and CI^" clearly indicates the biomineralization of the implant in vitro. The precipitation of these ions from the SBF solution towards the implant is attributed to the positive (Ca⁺²) and the negative (PO4 ³ and OH ) sites on the HAp coating. The spontaneous formation of an apatite layer on the HAp NP-based coating containing antibiotics suggests that the presence of the drug did not interfere with the formation of a new apatite layer. Thus, the combination between HAp and antibiotics such as Gs and Cip can guarantee superior osteoconductivity combined with antibacterial properties. This unique synergy has crucial role in determining the success of implantation by simultaneously promoting bone formation and reducing the risk of infections.

Antibacterial activity

Evaluation of the antibacterial activity of the drug-loaded HAp NPs coatings is crucial due to the fact that non-trivial conditions (high pH, 1 h at boiling point and high voltage) were used in order to synthesis and deposit the drug-loaded HAp NPs. The antibacterial activity was performed against Pseudomonas aeruginosa, a common bacteria, which is associated with bone infections. Cip and Gs are well-known antibiotics that have efficient antimicrobial activity against Gram-negative bacteria such as, Pseudomonas aeruginosa. The drug-loaded HAp NPs coatings were scraped from the titanium substrates and dispersed in PBS solution under sonication in order to release the antibiotics from the NPs. After centrifugation, the supernatants were tested against the Pseudomonas aeruginosa bacteria. Pristine HAp NPs coatings were used as negative control in order to show that neither HAp nor PBS has any antimicrobial activity. Pure Gs and Cip solutions (100 mg/mL in PBS) were used as positive control. Fig. 19 shows that the drug-loaded HAp NPs have efficient antibacterial activity compared to the positive control. These results confirmed that both the synthesis and the EPD does not impair the antibacterial properties of Cip and Gs. The average inhibition diameters of Cip and Gs-HAp NPs were 2.11±0.07, 2.53±0.05 cm, respectively. These results were similar to the positive control 2.26±0.08 and 2.73±0.1 cm for Gs and Cip solution, respectively. Hence, the performance of Gs and Cip is not impaired by the synthesis and EPD conditions, and therefore the antibiotics can be well-combined with HAp NPs to produce biocompatible coatings with excellent antibacterial activity.

Claims

CLAIMS:

1. A process for forming a film of calcium phosphate (CP) nanoparticles on at least a surface region of a substrate, the process comprising treating a dispersion of CP nanoparticles coated or associated with a plurality of pH-sensitive residues under conditions causing deposition of said nanoparticles on the surface region, wherein said nanoparticles optionally comprising at least one active or non-active material.

2. A process for forming a film of calcium phosphate (CP) nanoparticles on at least a surface region of a substrate, the process comprising forming a dispersion of CP nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of pH-sensitive residues, and subsequently treating said dispersion under acidic conditions or basic conditions to neutralize said pH-sensitive residues, to thereby cause deposition of said CP nanoparticles on the surface region, wherein said nanoparticles optionally comprising at least one active or non-active material.

3. The process according to claim 1 or 2, wherein the pH-sensitive residues are selected from carboxylates, amines, ammoniums, phosphates, phosphonates, phospites, sulfonates, sulfates and sulfinics, each of which being in a charged form.

4. The process according to claim 3, wherein the pH-sensitive residues are selected amongst negatively charged groups protonatable in presence of acid and positively charged groups having acidic proton removable in presence of a base.

5. The process according to any one of the preceding claims, wherein the deposition process comprises electrodeposition.

6. The process according to any one of claims 1 to 4, wherein the acidic or basic conditions involve application of a potential causing change in pH at the vicinity of the substrate, neutralization of the pH-sensitive residues and deposition on the substrate.

7. The process according to any one of the proceeding claims, the process comprising:

a. obtaining a dispersion of CP nanoparticles, optionally associated with at least one active or non-active material;

b. placing said dispersion in the vicinity of a substrate to be coated with said nanoparticles; and

c. modifying pH at the vicinity of said substrate to cause deposition of said nanoparticles on the substrate.

8. The process according to claim 7, wherein modifying pH comprises application of voltage.

9. The process according to claim 7, the process comprising electrodeposition of CP nanoparticles on a surface region of a substrate, the nanoparticles being coated or associated with a plurality of carboxylate residues, the process comprising contacting said substrate with a water dispersion of said nanoparticles under conditions permitting reduction in pH in the vicinity of the substrate and protonation of the residues.

10. The process according to claim 9, wherein the conditions permitting reduction in pH involve applying positive potential.

11. The process according to any one of the preceding claims, wherein the surface region is at least a surface region of a medical device.

12. The process according to any one of the preceding claims wherein the substrate is a metallic substrate.

13. The process according to any one of the preceding claims, wherein the substrate is conductive.

14. The process according to claim 12 or 13, wherein said metallic or conductive substrate comprises a metal selected from titanium, stainless steel, cobalt, chromium, gold and alloys thereof.

15. The process according to any one of the preceding claims, wherein said calcium phosphate (CP) is selected from hydroxy apatite (HAp), amorphous calcium phosphate and tricalcium phosphate.

16. The process according to any one of the preceding claims, wherein said calcium phosphate (CP) is hydroxy apatite (HAp).

17. The process according to any one of the preceding claims, wherein the CP nanoparticles are associated with at least one active agent.

18. The process according to claim 17, wherein the active agent is heat-sensitive.

19. The process according to claim 17 or 18, wherein the active agent is at least one drug or pharmaceutical.

20. A process for forming a film of nanoparticles on at least a surface region of a substrate, the nanoparticles comprising calcium phosphate (CP) and at least one heat- sensitive material and are further coated or associated with a plurality of pH- sensitive residues, the process comprising treating a dispersion of the nanoparticles in the vicinity of the substrate under conditions causing neutralization of the pH-sensitive residues and deposition of said nanoparticles on the surface region of the substrate.

21. The process according to claim 1, for forming a film of nanoparticles on at least a surface region of a substrate, the nanoparticles comprising calcium phosphate (CP) and at least one heat- sensitive material and are further coated or associated with a plurality of pH-sensitive residues, the process comprising treating a dispersion of the nanoparticles in the vicinity of the substrate under conditions causing neutralization of the pH-sensitive residues and deposition of said nanoparticles on the surface region of the substrate.

22. The process according to any one of claims 17 to 21, wherein the at least one active agent is selected from antibiotics, antibacterial agents, antifungal agents, antiviral agents, mitogenic growth factors, morphogenic growth factors, angiogenesis agents, anticancer agents, antiproliferative agents, anticlotting agents, antioxidants, analgesics, antiseptics, bioabsorbability/bioresorbability enhancers, bisphosphonates, calcitonins, chemotherapeutics, clotting agents, agents for treating pain, immune system boosters, immunosuppressants, immunomodulators, nutrients, statins, osteoclast inhibitors, antiinflammatory agents, osteogenic agents, and agents promoting osteointegration or osteoconduction.

23. The process according to claim 22, wherein the at least one active agent is an antibiotic.

24. The process according to claim 23, wherein the antibiotic is selected from cefazolin, cephradine, cefaclor, cephapirin, ceftizoxime, cefoperazone, cefotetan, cefutoxime, cefotaxime, cefadroxil, ceftazidime, cephalexin, cephalothin, cefamandole, cefoxitin, cefonicid, ceforanide, ceftriaxone, cefadroxil, cephradine, cefuroxime, ampicillin, amoxicillin, cyclacillin, ampicillin, penicillin G, penicillin V potassium, piperacillin, oxacillin, bacampicillin, cloxacillin, ticarcillin, azlocillin, carbenicillin, methicillin, nafcillin, erythromycin, tetracycline, doxycycline, minocycline, aztreonam, chloramphenicol, ciprofloxacin (Cip) hydrochloride, clindamycin, metronidazole, gentamicin, gentamicin sulfate (Gs), lincomycin, tobramycin, vancomycin, polymyxin B sulfate, colistimethate, colistin, azithromycin, augmentin, sulfamethoxazole, trimethoprim, and derivatives thereof.

25. The process according to claim 24, wherein the antibiotic is ciprofloxacin (Cip) hydrochloride, gentamicin or gentamicin sulfate (Gs).

26. The process according to claim 17, wherein the at least one agent is selected from an autograft materials, allograft materials, ceramic-based bone substitutes, and blends and mixtures thereof.

27. The process according to claim 17, wherein the at least one agent is selected from corticosteroids, oxysterols, compounds that upregulate intracellular cAMP, and compounds that impact the HMG coA reductase pathway and blends and mixtures thereof.

28. The process according to claim 17, wherein the at least one active agent is a corticosteroid selected from budesonide, fluticasone propionate, fluoromethalone, halcinonide, clobetasol propionate, and blends and mixtures thereof.

29. The process according to any one of the preceding claims being carried at room temperature.

30. A film comprising CP, the film being formed by a process according to any one of claims 1 to 29.

31. The film according to claim 30, further comprising at least one active agent, being optionally heat-sensitive.

32. A film nanoparticles comprising CP and at least one heat- sensitive material, the film being on a surface region of a substrate.

33. The film according to claim 32, being formed by a process according to any one of claims 1 to 29.

34. A medical device comprising a film according to any one of claims 30 to 33.

35. A film according to any one of claims 30 to 33, for promoting osteointegration and osteoconduction.

36. A process for promoting osteointegration or osteoconduction properties to a surface region of an implantable medical device, the process comprising forming a film of calcium phosphate (CP) nanoparticles on the surface by causing electrodeposition of said nanoparticles onto the surface.

37. The process according to claim 36, the process comprising forming a dispersion of nanoparticles, at least a surface region of the nanoparticles being coated or associated with a plurality of pH-sensitive residues, and subsequently treating said dispersion under acidic conditions or basic conditions, to cause neutralization of the pH-sensitive residues and deposition of said nanoparticles on the surface region of the medical device.

38. The process according to claim 37, the process comprising electrodeposition of calcium phosphate (CP) nanoparticles on the surface region of the medical device, the nanoparticles being coated or associated with a plurality of pH-sensitive residues, the process comprising contacting said substrate with a water dispersion of said nanoparticles under conditions permitting reduction in pH in the vicinity of the substrate and protonation of the residues.

39. The process according to any one of claims 36 to 38, wherein the calcium phosphate (CP) is selected from hydroxy apatite (HAp), amorphous calcium phosphate and tricalcium phosphate.

40. The process according to claim 39, wherein said calcium phosphates (CP) is hydroxyapatite (HAp).