Sequential Targeting of Retinoblastoma and DNA Synthesis Pathways Is a Therapeutic Strategy for Sarcomas That Can Be Monitored in Real Time.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. SIGNIFICANCE: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.

Low-molecular-weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer.

Low-molecular-weight cyclin E (LMW-E) is an N-terminus deleted (40 amino acid) form of cyclin E detected in breast cancer, but not in normal cells or tissues. LMW-E overexpression predicts poor survival in breast cancer patients independent of tumor proliferation rate, but the oncogenic mechanism of LMW-E and its unique function(s) independent of full-length cyclin E (FL-cycE) remain unclear. In the current study, we found LMW-E was associated with genomic instability in early-stage breast tumors (n = 725) and promoted genomic instability in human mammary epithelial cells (hMECs). Mechanistically, FL-cycE overexpression inhibited the proliferation of hMECs by replication stress and DNA damage accumulation, but LMW-E facilitated replication stress tolerance by upregulating DNA replication and damage repair. Specifically, LMW-E interacted with chromatin and upregulated the loading of minichromosome maintenance complex proteins (MCMs) in a CDC6 dependent manner and promoted DNA repair in a RAD51- and C17orf53-dependent manner. Targeting the ATR-CHK1-RAD51 pathway with ATR inhibitor (ceralasertib), CHK1 inhibitor (rabusertib), or RAD51 inhibitor (B02) significantly decreased the viability of LMW-E-overexpressing hMECs and breast cancer cells. Collectively, our findings delineate a novel role for LMW-E in tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E-overexpressing breast cancers.

Re-engineered imaging agent using biomimetic approaches.

Recent progress in biomedical technology, the clinical bioimaging, has a greater impact on the diagnosis, treatment, and prevention of disease, especially by early intervention and precise therapy. Varieties of organic and inorganic materials either in the form of small molecules or nano-sized materials have been engineered as a contrast agent (CA) to enhance image resolution among different tissues for the detection of abnormalities such as cancer and vascular occlusion. Among different innovative imaging agents, contrast agents coupled with biologically derived endogenous platform shows the promising application in the biomedical field, including drug delivery and bioimaging. Strategy using biocomponents such as cells or products of cells as a delivery system predominantly reduces the toxic behavior of its cargo, as these systems reduce non-specific distribution by navigating its cargo toward the targeted location. The hypothesis is that depending on the original biological role of the naïve cell, the contrast agents carried by such a system can provide corresponding natural designated behavior. Therefore, by combining properties of conventional synthetic molecules and nanomaterials with endogenous cell body, new solutions in the field of bioimaging to overcome biological barriers have been offered as innovative bioengineering. In this review, we will discuss the engineering of cell and cell-derived components as a delivery system for various contrast agents to achieve clinically relevant contrast for diagnosis and study underlining mechanism of disease progression. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Strategic reconstruction of macrophage-derived extracellular vesicles as a magnetic resonance imaging contrast agent.

A contrast agent (CA) in magnetic resonance imaging (MRI) is now an essential add-on to obtain high-quality contrast-enhanced anatomical images for disease diagnosis and monitoring the treatment response. However, the rapid elimination of CAs by the immune system and excretion by the renal route has limited its application. As a result, the CA dose for effective contrast is ever-increasing, resulting in toxic side effects such as gadolinium (Gd) related nephrogenic systemic fibrosis (NSF) toxicity. Considering the widespread application of Gd-based CAs, it is now very important to revisit their formulation in order to improve their local concentration and minimize their dose while achieving clinical goals. Therefore, we have adapted a unique strategy to maximize Gd delivery to the target site using macrophage cell-derived extracellular vesicles (EVs) reconstructed with a Gd-conjugated liposomal system herein called gadolinium infused hybrid EVs (Gd-HEVs). We hypothesize that Gd-HEVs, owing to the presence of immune cell-derived EV protein cargo, can effectively disguise themselves as a biological entity, prolong the retention time for contrast enhancement, and show tumor specificity. Incorporation of Gd into nanoformulations can enhance the longitudinal relaxivity r1 by reducing the tumbling rate of paramagnetic metal complexes. Here, Gd-HEVs showed a higher r1 relaxivity of 9.86 mM-1 s-1 compared to 3.98 mM-1 s-1 of Magnevist® at an equivalent Gd concentration, when measured by clinical 3T MRI. This will allow us to reduce the clinically used Gd concentration about three-fold while maintaining contrast in the clinical window thereby supporting our hypothesis. Furthermore, Gd-HEVs showed a preferential cellular interaction and accumulation towards cancer cells compared to non-cancer cells, both in vitro and in vivo. More importantly, Gd-HEVs showed excellent contrast enhancement in the blood vasculature with a higher retention time compared to its counterpart, Magnevist®. Our study successfully showed that the incorporation of Gd in the EV framework can help to enhance the contrast ability, and therefore it can be a platform technology for the development of safer MRI contrast agents.

Erythrocyte membrane concealed paramagnetic polymeric nanoparticle for contrast-enhanced magnetic resonance imaging.

Recent progress in bioimaging nanotechnology has a great impact on the diagnosis, treatment, and prevention of diseases by enabling early intervention. Among different types of bioimaging modalities, contrast-enhanced magnetic resonance imaging using paramagnetic gadolinium-based molecular contrast agents (GBCAs) are most commonly used in clinic. However, molecular GBCAs distribute rapidly between plasma and interstitial spaces with short half-lives limiting its clinical impacts. To improve the properties of GBCAs, herein an effort has been put forth by incorporating GBCA into nanoscale system mimicking the property of red blood cell (RBC) that could facilitate contrast enhancement and prolong intraluminal retention in the body. The proposed nanoconstruct is made up of polymeric-core labeled with lipid conjugated GBCA followed by the imprint of the RBC membrane concealment layer to enhance stability and biocompatibility. Meanwhile, the confinement strategy of GBCA was implemented to accelerate magnetic properties of nanoconstruct providing longitudinal-relaxivity (r1) to 12.78 ± 0.29 (mM s)-1. Such improvement in r1 was further confirmed by enhanced contrast in the vascular angiography of the murine model. Given higher colloidal stability and tunable magnetic properties, nanoconstruct proposed herein is a promising platform technology for the applications where enhanced plasma residence time and magnetic properties are necessary for diagnosis and therapy.

pH-responsive cationic liposome for endosomal escape mediated drug delivery.

Endosomal degradation of the nanoparticle is one of the major biological barriers associated with the drug delivery system. Nanoparticles are internalized in the cell via different endocytosis pathways, where they are first delivered to early endosomes which mature to the late endosome and to the lysosome. During this journey, NP encounters a harsh chemical environment resulting in the degradation of NP and its content. This process is collectively called as intracellular defenses against foreign materials. Therefore, to avoid this degradative fate, the endosomal escape technique has been explored following membrane fusion or membrane destabilization mechanisms. However, these methods are limited to the application due to non-specific membrane fusion. To overcome this limitation, we have designed pH-responsive liposome made up of 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-liposome) in which the cationic nitrogen of the ammonium moiety occupies only ∼2.5 % of the molecule. Such a small percentage of the cationic moiety is sufficient enough to exhibit pH-responsive properties while maintaining the biocompatibility of the DC-liposome. DC-liposome showed pH-dependent cationic properties due to the protonation of DC-moiety at acidic pH. The fluorescence-based experiment confirmed pH-dependent fusogenic properties of DC-liposome. Furthermore, the endosomal colocalization study revealed higher localization of DC-liposome in the early endosome compared to that of the late endosome, suggesting possible endosomal escape. Elevated cationic and fusogenic properties of DC-liposome at acidic pH can mediate membrane fusion with anionic endosomal membrane via electrostatic interaction, thereby causing endosomal escape. Moreover, doxorubicin-loaded DC-liposome showed higher cytotoxicity than that of free doxorubicin further supporting our clam of endosomal escape. These findings suggest the potential of DC-liposome to break the endosomal barriers to enhance the therapeutic efficacy thereby guiding us in design consideration in the field of stimuli-responsive delivery agents.

Biodistribution of gadolinium- and near infrared-labeled human umbilical cord mesenchymal stromal cell-derived exosomes in tumor bearing mice.

We speculate that exosomes derived from human umbilical cord mesenchymal stromal cells (HUC-MSCs) will accumulate within tumors and have the potential for both tumor location or drug delivery. Methods: To determine proof of concept, HUC-MSC exosomes were labeled with an MRI contrast agent, gadolinium, or a near infrared dye. Exosome accumulation within ectopic osteosarcoma tumor-bearing mice was determined by 14.1 T MRI or bioimaging over 24-48 h after injection. In vitro studies examine the accumulation and physiological effect of exosomes on human and mouse osteosarcoma cell lines by MTT assay, confocal microscopy, and flow cytometry. Results: Systemic HUC-MSC exosomes accumulated continuously in tumor over a 24-48 h post-injection period. In contrast, synthetic lipid nanoparticles accumulate in tumor only for the first 3 h post-injection. Conclusion: These results suggest that HUC-MSCs exosomes accumulate within human or mouse osteosarcoma cells in vitro and in vivo over a 24 to 48 h after infusion.

Macrophage-derived exosome-mimetic hybrid vesicles for tumor targeted drug delivery.

Extracellular vesicles (EVs) are phospholipid and protein constructs which are continuously secreted by cells in the form of smaller (30-200 nm) and larger (micron size) particles. While all of these vesicles are called as EVs, the smaller size are normally called as exosomes. Small EVs (sEVs) have now been explored as a potential candidate in therapeutics delivery owing to their endogenous functionality, intrinsic targeting property, and ability to cooperate with a host defense mechanism. Considering these potentials, we hypothesize that immune cell-derived sEVs can mimic immune cell to target cancer. However, different sEVs isolation technique reported poor yield and loss of functional properties. To solve this problem, herein we hybridized sEVs with synthetic liposome to engineer vesicles with size less than 200 nm to mimic the size of exosome and named as hybrid exosome (HE). To achieve this goal, sEVs from mouse macrophage was hybridized with synthetic liposome to engineer HE. The fluorescence-based experiment confirmed the successful hybridization process yielding HE with the size of 177 ±â€¯21 nm. Major protein analysis from Blot techniques reveal the presence of EV marker proteins CD81, CD63, and CD9. Differential cellular interaction of HE was observed when treated with normal and cancerous cells thereby supporting our hypothesis. Moreover, a water-soluble doxorubicin was loaded in HE. Drug-loaded HE showed enhanced toxicity against cancer cells and pH-sensitive drug release in acidic condition, benefiting drug delivery to acidic cancer environment. These results suggest that the engineered HE would be an exciting platform for tumor-targeted drug delivery. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) are phospholipid and protein constructs which are continuously secreted by cells in the human body. These vesicles can efficiently deliver their parental biomolecules to the recipient cells and assist in intracellular communication without a direct cell-to-cell contact. Moreover, they have the ability to perform some of the molecular task similar to that of its parent cells. For example, exosome derived from immune cells can seek for diseased and/or inflammatory cells by reading the cell surface proteins. However, different EVs isolation techniques reported poor yield and loss of functional properties. Therefore, to overcome this limitation, we herein propose to re-engineer immuno-exosome with a synthetic liposome as a refined biomimetic nanostructure for the delivery of doxorubicin (clinical drug) for breast cancer treatment.

Biomimetic surface modification of discoidal polymeric particles.

The rationale for the design of drug delivery nanoparticles is traditionally based on co-solvent self-assembly following bottom-up approaches or in combination with top-down approaches leading to tailored physiochemical properties to regulate biological responses. However, the optimal design and control of material properties to achieve specific biological responses remain the central challenge in drug delivery research. Considering this goal, we herein designed discoidal polymeric particles (DPPs) whose surfaces are re-engineered with isolated red blood cell (RBC) membranes to tailor their pharmacokinetics. The RBC membrane-coated DPPs (RBC-DPPs) were found to be biocompatible in cell-based in vitro experiments and exhibited extended blood circulation half-life. They also demonstrated unique kinetics at later time points in a mouse model compared to that of bare DPPs. Our results suggested that the incorporation of biomimicry would enable the biomimetic particles to cooperate with systems in the body such as cells and biomolecules to achieve specific biomedical goals.

The influence of polyethylene glycol passivation on the surface plasmon resonance induced photothermal properties of gold nanorods.

Gold nanorods (AuNRs) possess unique photothermal properties due to their strong plasmonic absorption in the near-infrared region of the electromagnetic spectrum. They have been explored widely as an alternative or a complement to chemotherapy in cancer treatment. However, the use of AuNRs as an injectable medicine is greatly hindered by their stability in biological media. Therefore, studies have been focused on improving the stability of AuNRs by introducing biocompatible surface functionalizations such as polyethylene glycol (PEG) coatings. However, these coatings can affect heat conduction and alter their photothermal behavior. Herein, we studied how functionalization of AuNRs with PEG chains of different molecular weights determined the temperature distribution of suspensions under near-infrared irradiation, cell uptake in vitro, and hyperthermia-induced cytotoxicity. Thermogravimetric analysis of the PEG-conjugated AuNRs exhibited slightly different PEG mass fractions of 12.0%, 12.7%, and 18.5% for PEG chains with molecular weights of 2, 5, and 10 kDa, respectively, implying distinct structures for PEG brushes. When exposed to near-infrared radiation, we found greater temperatures and temperature gradients for longer PEG chains, while rapid aggregation was observed in unmodified (raw) AuNRs. The effect of the PEG coating on heat transport was investigated using molecular dynamics simulations, which revealed the atomic scale structure of the PEG brushes and demonstrated lower thermal conductivity for PEG-coated AuNRs than for unmodified AuNRs. We also characterized the uptake of the AuNRs into mouse melanoma cells in vitro and determined their ability to kill these cells when subjected to near-infrared radiation. For all PEG-coated AuNRs, exposure to 10 s of near-infrared radiation significantly reduced cell viability relative to unirradiated controls, with this viability further decreasing with increasing AuNR doses, indicating potential phototherapeutic effects. The 5 kDa PEG coating appeared to yield the best performance, yielding significant phototoxicity at even the lowest dose considered (0.5 µg mL-1), while also exhibiting high colloidal stability, which could help in rational design consideration of AuNRs for NIR induced photothermal therapy.

Natural killer cell membrane infused biomimetic liposomes for targeted tumor therapy.

Therapeutic efficacy of a systemic drug delivery largely depends on the targeting design of the delivery system, which tackles with circulatory traffic and prevents the nonspecific distribution of the drug in the wide range of vital organs. A drawing attention has been given to a biomimetic cloaking of the synthetic drug delivery nanoparticle using mammalian cell-ghosts, which has shown the installment of the biological complexity of the original cells thereby acting as naïve cells, to precisely delivery drug to the intended target. Align towards this direction; we developed a membrane camouflage fusogenic liposomal delivery system "NKsome" for targeted tumor therapy using Natural Killer (NK) cell-ghost, which naturally undergoes immunosurveillance of diseased/stress cells. The engineered NKsome shows successful retention of NK cell membrane-associated targeting protein on its surface. With its excellent biocompatibility, NKsome shows a higher affinity towards cancer than normal cells as demonstrated by in vitro flow-passage assay, and exhibits enhanced tumor homing efficiency in-vivo with an extended plasma residence time of 18 h. Moreover, the therapeutic potential of doxorubicin-loaded NKsome shows promising antitumor activity in vivo against MCF-7 induced tumor model. Overall results illustrate the therapeutic advantages of NK cell biomimicry capable of communicating like immune cells for cooperative drug delivery.

Nano-confinement-driven enhanced magnetic relaxivity of SPIONs for targeted tumor bioimaging.

Superparamagnetic iron oxide nanoparticles (SPIONs) are highly biocompatible and have a versatile synthetic technique based on coprecipitation, reduction-precipitation, and hydrothermal methods, where Fe3+ and Fe2+ react in aqueous solutions; both these ions are present in our body and have clear metabolic pathways; therefore, they have attracted extensive research interest and development in the field of diagnostic imaging and therapy. However, most SPION-based clinical diagnostic contrast agents are discontinued due to severe pain, low transverse magnetic relaxivity range of 80-180 mM-1 s-1, shorter circulation half-life, and lack of disease specificity. Therefore, in this study, we engineered a bone cancer-targeted hybrid nanoconstruct (HNC) with a high transverse magnetic relaxivity of 625 mM-1 s-1, which was significantly higher than that of clinical contrast agents. The engineered HNC is peripherally decorated with a bone-seeking agent, alendronic acid-conjugated phospholipid, exhibiting a hydrodynamic size of 80 nm with a negative surface potential, -35 mV. The interior skeleton of the HNC is composed of biodegradable and biocompatible poly(l-lactic-co-glycolic acid) (PLGA), in which 5 nm SPIONs are confined. We have successfully tuned the distance between the confined SPIONs from 0.5 to 4 nm, as revealed by transmission electron microscopy (TEM) images and magnetic resonance image (MRI) phantoms. This cluster confinement dramatically enhances magnetic relaxivity possibly due to the increase in net local magnetization due to proximal field inhomogeneity. In an in vitro examination, 80% of HNC is found to bind with hydroxyapatite (HAp), which when characterized by TEM shows a painting of SPIONs over a HAp crystal. HNC is found to accumulate in mouse osteosarcoma tumor (K7M2 tumor model); both MRI and histological examination of the tumor show the potential of HNC as targeting agents for diagnosis of tumor in the bone.

Impact of cell adhesion and migration on nanoparticle uptake and cellular toxicity.

In vitro cell-nanoparticle (NP) studies involve exposure of NPs onto the monolayer cells growing at the bottom of a culture plate, and assumed that the NPs evenly distributed for a dose-responsive effect. However, only a few proportion of the administered dose reaches the cells depending on their size, shape, surface, and density. Often the amount incubated (administered dose) is misled as a responsive dose. Herein, we proposed a cell adhesion-migration (CAM) strategy, where cells incubated with the NP coated cell culture substrate to maximize the cell-NP interaction and investigated the physiological properties of the cells. In the present study, cell adhesion and migration pattern of human breast cancer cell (MCF-7) and mouse melanoma cell (B16-F10) on cell culture substrate decorated with toxic (cetyltrimethylammonium bromide, CTAB) and biocompatible (poly (sodium 4-styrenesulphonate), PSS) gold nanoparticles (AuNPs) of different sizes (5 and 40nm) were investigated and evaluated for cellular uptake efficiency, proliferation, and toxicity. Results showed enhanced cell adhesion, migration, and nanoparticle uptake only on biocompatible PSS coated AuNP, irrespective of its size. Whereas, cytotoxic NP shows retard proliferation with reduced cellular uptake efficiency. Considering the importance of cell adhesion and migration on cellular uptake and cytotoxicity assessment of nanoparticle, CAM strategy would hold great promises in cell-NP interaction studies.

A review on nanoparticle-based technologies for biodetoxification.

Nanotechnology has gained significant penetration to different fields of medicine including drug delivery, disease interrogation, targeting and bio-imaging. In recent years, efforts have been put forth to assess the use of this technology in biodetoxification. In this review, we will discuss the current status of nanostructured biomaterials/nanoparticle (NP)-based technologies as a candidate biodetoxifying agent. Patient hospitalization due to illicit drug consumption, suicidal attempts and accidental toxin exposure are major challenges in the medical field. Overdoses of drugs/toxic chemicals or exposure to bacterial toxins or poisons are conventionally treated by voiding the stomach, administering activated charcoal or by using specific antidotes, if the toxin is known. Because of the limitations of these methods for safe and effective detoxification, advancements in nanotechnology may offer novel ways in intoxication support by using nanostructured biomaterials, such as liposomes, micellar nanocarriers, liquid crystalline nanoassemblies and ligand-based NPs.

Membrane Fusion-Mediated Gold Nanoplating of Red Blood Cell: A Bioengineered CT-Contrast Agent.

Red blood cells (RBCs) are the natural resident of the vascular lumen, therefore delivery of any agents within the vascular lumen could benefit by unique natural transporting features of RBCs. RBCs continuously circulate for ∼100 days before being sequestered in the spleen, they only extravasate at sites of vascular hemorrhage. Taking advantages of these features, we engineered RBC as a carrier in order to design a unique delivery system capable of delivering X-ray computed tomography (CT) contrast agents, gold nanoparticles (AuNPs), thereby acting as CT-contrast agent. A strategic membrane fusion technique was used to engineer the surface of RBC with gold nanoparticles in this in vitro study without altering its shape, size, and surface properties.

Engineered Nanomedicine with Alendronic Acid Corona Improves Targeting to Osteosarcoma.

We engineered nanomedicine with the stealth corona made up of densely packed bone seeking ligand, alendronic acid. In a typical nanoconstruct, alendronic acid is conjugated with hydrophilic head moiety of phospholipid that has an ability to self-assemble with hydrophobic polymeric core through its hydrophobic long carbon-chain. Proposed nanomedicine has three distinct compartments namely; poly(l-lactic-co-glycolic acid) polymeric core acting as a drug reservoir and skeleton of the nanoconstruct, phospholipid monolayer covers the core acting as a diffusion barrier, and a densely packed alendronic acid corona acting as a stabilizer and targeting moiety. Thus engineered nanomedicine attain spherical entity with ~90 ± 6 nm having negative zeta potential, -37.7 ± 2 mV, and has an ability to load 7 ± 0.3 wt% of doxorubicin. In-vitro bone targeting efficiency of nanomedicine was studied using hydroxyapatite crystals as a bone model, and found significant accumulation of nanoparticle in the crystals. Moreover, cellular internalization studies with mouse osteosarcoma confirm the selectivity of nanomedicine when compared to its internalization in non-targeted mouse melanoma. This nanomedicine shows prolong stability in serum and deliver the drug into the cell exhibiting an IC50 of 3.7 µM. Given the strong interacting property of alendronic acid with bone, the proposed nanomedicine hold promises in delivering drug to bone microenvironment.

Synthesis and Characterization of Biomimetic Hydroxyapatite Nanoconstruct Using Chemical Gradient across Lipid Bilayer.

In this study, we synthesized biomimetic hydroxyapatite nanoconstruct (nanosized hydroxyapatite, NHAp) using a double emulsion technique combined with a chemical gradient across a lipid bilayer for surface modification of a titanium (Ti) implant. The synthesized NHAp was characterized by dynamic light scattering, X-ray diffraction, transmission electron microscopy, and Fourier transform infrared (FTIR) spectroscopy, and it was further tested for its biocompatibility and in vitro proliferation efficacy using normal human osteoblasts (NHOst). The results showed that the synthesized NHAp had a hydrodynamic diameter of ∼200 nm with high aqueous stability. The chemistry of the NHAp was confirmed by FTIR spectroscopic analysis. Typical FTIR vibrational bands corresponding to the phosphate group (PO4(3-)) present in hydroxyapatite (HAp) were observed at 670, 960, and 1000 cm(-1). A broad band at 3500 cm(-1) confirmed the presence of a structural -OH group in the NHAp. Powder X-ray crystallographic diffraction further confirmed the formation of NHAp with characteristic reflections in (002), (211), (130), and (213) planes at respective 2θ degrees. These reflection planes are similar to those of typical HAp crystallized toward (002) and (211) crystallographic planes. The mechanism of the formation of NHAp was studied using the fluorescence resonance energy transfer (FRET) technique. The FRET study showed the fluorescent recovery of a donor fluorophore and the mechanism of the insertion of lipids into nanodroplets obtained from the first water-in-oil (w/o) emulsion during the formation of the second oil-in-water (o/w) emulsion. With these confirmations, we further studied NHOst cell proliferation on a Ti surface. When NHOst were cultured on the Ti surface coated with the NHAp, a distinct proliferation pattern and cell-cell communication via cytoplasmic extension on the substrate surface were observed. In contrast, a bare Ti surface showed diminished cell size with minimal adherence. This result indicates that our NHAp covered with a phospholipid bilayer provides a proper environment essential for cell adhesion, which is especially important for bone implants, and the inclusion of NHAp on the Ti substrate would be an effective support for long-term sustainability of implants.