Modified nanodelivery system and method for enhanced in vivo medical and preclinical imaging

Abstract

A lipid-, polymer-, and metal-based system of modified nanostructures of active biomedical and pharmaceutical agents used for in vivo (whole body/organ or tissue-specific) imaging. The modified nanostructure system involves various combinations of excipients (lipids, oils, surfactant, polymers, metals, carbon, nanotubes, etc.) in a formulation that allows a user to: (1) sustain the bioluminescent, fluorescent, or contrast signal for a longer period than conventional systems without repetitive administration (e.g., nanostructure system of luciferin), (2) target specific sites of interest (e.g., organ, tissue, receptors, proteins, etc.) for enhanced imaging of the targeted site (e.g. nanostructure system of XenoLight DIR with CREKA allows imaging of tumor vasculature), and (3) increase bioluminescent, fluorescent, or contrast signal flux.

Claims

1. A nanoparticle delivery system for intravenously delivering an active substance or agent to a target within a subject for medical and preclinical imaging, comprising: at least one nanoparticle carrier comprising about 700 mg of at least one solid phase lipid, about 330 mg of at least one liquid phase lipid or oil, and about 480 μl of at least one surfactant; wherein the at least one solid phase lipid is selected from the group consisting of propylene glycol palmitostearate, glyceryl palmitostearate, and combinations thereof; wherein the at least one liquid phase lipid or oil is selected from the group consisting of caprylic/capric triglycerides, medium chain triglycerides, diethylene glycol monoethyl ether and combinations thereof; wherein the at least one surfactant is selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate and a combination thereof; a luciferin encapsulated within the at least one nanoparticle carrier; an active pharmaceutical agent encapsulated within the at least one nanoparticle carrier; and a target-honing molecule conjugated to a surface of the at least one nanoparticle carrier; wherein biofluorescence of the luciferin occurs as a steady release for up to about 4 hours after intravenous administration of the nanoparticle delivery system to the subject; wherein the biofluorescence of the luciferin is detectable in the subject for at least 24 hours after intravenous administration of the nanoparticle delivery system to the subject; wherein release rate of the luciferin is about 50% at 24 hours after intravenous administration with at least 92% encapsulation efficiency; wherein the nanoparticle delivery system is less than about 200 nm in size; wherein the nanoparticle delivery system is formed by hot melt homogenization followed by high pressure homogenization.

2. The nanoparticle delivery system of claim 1, wherein the at least one liquid phase lipid is the caprylic/capric triglyceride.

3. The nanoparticle delivery system of claim 1, wherein the pharmaceutical agent is 1,1-bis(3′ indolyl)-1-(p-biphenyl)methane (DIM-C-pPhC.sub.6H.sub.5).

4. The nanoparticle delivery system of claim 1, wherein the target-honing molecule targets a tumor in the subject.

5. The nanoparticle delivery system of claim 4, wherein the target-honing molecule is a CREKA peptide.

6. The nanoparticle delivery system of claim 1, wherein nanoparticle delivery system is generated by the process consisting essentially of: dissolving the bioimaging agent in an organic solvent to form an organic phase solution; mixing the at least one solid phase lipid and the at least one liquid phase lipid to form a lipid solution; mixing the organic phase solution with the lipid solution to form a lipid phase solution; heating the lipid phase solution to remove the organic solvent; mixing water and a surfactant to form an aqueous phase solution; mixing the lipid phase solution with the aqueous phase solution to form a mixture; performing high pressure homogenization on the mixture wherein the at least one nanoparticle carrier encapsulating the bioimaging agent is generated; and conjugating the target-honing agent to the surface of the at least one nanoparticle carrier.

7. The nanoparticle delivery system of claim 1, further comprising a nickel chelating compound used as a spacer to conjugate the target-honing molecule.

8. A nanoparticle delivery system for intravenously delivering an active substance or agent to a target within a subject for medical and preclinical imaging, consisting essentially of: at least one nanoparticle carrier comprising about 700 mg of a solid phase lipid, about 330 mg of a liquid phase lipid or oil, and about 480 μl of at least one surfactant; wherein the solid phase lipid is propylene glycol palmitostearate; wherein the liquid phase lipid or oil is a caprylic/capric triglyceride; wherein the at least one surfactant is selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate and a combination thereof; a luciferin encapsulated within the at least one nanoparticle carrier; a target-honing molecule conjugated to a surface of the at least one nanoparticle carrier; and a nickel chelating compound used as a spacer to conjugate the target-honing molecule; wherein biofluorescence of the luciferin occurs as a steady release for up to about 4 hours after intravenous administration of the nanoparticle delivery system to the subject; wherein the biofluorescence of the luciferin is detectable in the subject for at least 24 hours after intravenous administration of the nanoparticle delivery system to the subject; wherein release rate of the luciferin is about 50% at 24 hours after intravenous administration with at least 92% encapsulation efficiency; wherein the nanoparticle delivery system is less than 200 nm in size; wherein the nanoparticle delivery system is formed by hot melt homogenization followed by high pressure homogenization.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

(2) FIG. 1 is a schematic illustrating preparation of modified nanoparticles according to an embodiment of the current invention.

(3) FIG. 2A is an in vivo image of an A549 and H460 lung cancer cell tumor bearing mouse in in vivo imaging system and spectrally unmixed image of vasculature.

(4) FIG. 2B is an in vivo image of FIG. 2A with PCNCs-Di.

(5) FIG. 2C is an in vivo image of FIG. 2A with NCs-Di.

(6) FIG. 3 is a graphical illustration showing that all three modified nanoparticles (Nano-Luc1, 2, and 3) produced equivalent level of luciferase signals. The luciferase signals were brightest at 30-60 minutes after injection of the Nano-Lucs. Nano-Luc1 and Nano-Luc3 prolonged the luciferase signal as compared with free luciferin.

(7) FIG. 4 is a graphical illustration showing that all three Nano-Lucs displayed different luciferase kinetics. Nano-Luc1 and Nano-Luc2 showed peak luciferase signal at 40-50 minutes. Nano-Luc3 showed continuous increase of luciferase signal over 60 minutes.

(8) FIG. 5A depicts the effect of lipid- and oil-type on loading efficiency.

(9) FIG. 5B depicts the effect of lipid- and oil-type on entrapment efficiency.

(10) FIG. 5C depicts the effect of lipid- and oil-type on 24-hour release rate.

(11) FIG. 5D depicts the effect of lipid- and oil-type in the RSM.

(12) FIG. 6A is response surface plots showing the effect of different concentration of lipid and oil on release rate, loading efficiency and entrapment efficiency of Nano-Luc.

(13) FIG. 6B is contour plots showing the effect of different concentration of lipid and oil on release rate, loading efficiency and entrapment efficiency of Nano-Luc.

(14) FIG. 7A is a response surface plot of desirability function.

(15) FIG. 7B is a contour plot of desirability function.

(16) FIG. 8A depicts differential scanning calorimetry of luciferin.

(17) FIG. 8B depicts differential scanning calorimetry of geleol.

(18) FIG. 8C depicts differential scanning calorimetry of Nano-Luc (formulation 1).

(19) FIG. 8D depicts differential scanning calorimetry of precirol.

(20) FIG. 8E depicts differential scanning calorimetry of Nano-Luc (formulation 2).

(21) FIG. 8F depicts differential scanning calorimetry of myglyol.

(22) FIG. 9 is a graph of log (% luciferin recovery) vs. time for the effect of temperature (30° C., 40° C. and 50° C.) on Nano-Luc stability.

(23) FIG. 10A depicts Nano-Luc tumor imaging in mice with subcutaneous 4T1-luc2 tumors using 150 mg/kg luciferin equivalent Nano-Luc by IP.

(24) FIG. 10B depicts Nano-Luc tumor imaging in mice with orthotopic 4T1-luc2 tumors using 150 mg/kg luciferin equivalent Nano-Luc by IP.

(25) FIG. 11A depicts Nano-Luc tumor imaging in mice with subcutaneous H460-luc2 tumors using 150 mg/kg luciferin equivalent Nano-Luc by IV.

(26) FIG. 11B depicts Nano-Luc tumor imaging in mice with orthotopic 4T1-luc2 tumors using 150 mg/kg luciferin equivalent Nano-Luc by IV.

(27) FIG. 11C depicts Nano-Luc tumor imaging in mice with mb231-luc2 metastatic tumors using 150 mg/kg luciferin equivalent Nano-Luc by IV.

(28) FIG. 12A is a graphical illustration showing in vivo kinetics of Nano-Luc compared to free luciferin, total flux of bioluminescence vs. time plot following IP injection of Nano-Luc and free luciferin.

(29) FIG. 12B is a graphical illustration showing in vivo kinetics of Nano-Luc compared to free luciferin, normalized to peak signal flux vs. time plot following subcutaneous injection of Nano-Luc and free luciferin.

(30) FIG. 12C is a graphical illustration showing in vivo kinetics of Nano-Luc compared to free luciferin, total flux of bioluminescence vs. time plot following IV injection of Nano-Luc and free luciferin.

(31) FIG. 13A is a graphical illustration showing in vivo kinetics of NanoLuc-DiR compared to free luciferin, normalized to peak signal flux vs. time plot following subcutaneous injection of NanoLuc-DiR and free luciferin.

(32) FIG. 13B is a graphical illustration showing in vivo kinetics of NanoLuc-DiR compared to free luciferin, total flux of fluorescence vs. time plot following dose of NanoLuc-DiR.

(33) FIG. 14 is a graphical illustration showing the release rate profile of luciferin from Nano-Luc in vitro.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(34) In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part thereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention.

(35) The present invention relates to a method of delivery of modified nanostructures of active biomedical and pharmaceutical agent(s) for longer sustained bioluminescent, fluorescent, or contrast signals and increased signal flux at specific sites of interest on the body. The invention is described herein in detail using the terms defined below unless otherwise specified.

(36) The terms “nanostructured carrier nanoparticles” (NCN or NCNs) and “nanostructured carrier” (NC or NCs) are used interchangeably and are meant to describe the plurality of lipid, metal, polymers, or carbon nanotube carrier nanoparticles forming the nanostructure. The lipid, metal, polymer, or carbon nanotube carrier nanoparticles in the nanostructure are produced using blends of solid and liquid (lipids/oils) ingredients. To obtain blends for the particles in the nanostructure, solids are mixed with liquids in a desired ratio. The blends obtained are solid at body temperature. These NCNs can encapsulate active substances; NCNs can be produced by known hot or cold high pressure homogenization techniques.

(37) Exemplary oil-based or lipidic medium carriers for the NCN are mono-, di- and triglycerides or mixtures thereof, and nickel chelating compounds. In a preferred embodiment, the oil-based or lipidic medium carrier may be COMPRITOL 888 ATO brand, MIGLYOL 812 brand, and DOGS-NTA-Ni chelating lipid and the like.

(38) A nanoparticle or particulate of this invention has an active substance encapsulated within the nanoparticles forming the nanostructure or carrier system. Methods of preparing nanoparticles that include an active substance encapsulated within are known to those skilled in the art.

(39) As an example used herein, “encapsulated within” means the active substance is contained substantially inside the NCN or nanoparticle.

(40) Modifying a surface of the nanostructured carrier nanoparticles refers to the method of coating the outside of the surface of the nanoparticles with a drug delivery enhancer. More specifically, the surface is modified by engraftment intern coating of the drug delivery enhancer onto the nanoparticle, for example coating a CREKA peptide onto the nanoparticle surface. The engraftment can be accomplished by using, for example, DOGS-NTA-Ni chelating/spacer (e.g., lipid), wherein the DOGS (portion of the spacer, e.g., lipid portion) is embedded in the preformed nanoparticle, and the NTA-Ni portion/tail of the spacer is projected outside of the nanoparticle surface such that the 6-histidine on the peptide bonds strongly to NTA-Ni portion/tail to form a nanoparticle with a NTA-Ni-6Histidine-CREKA surface. It is well-known in the field that histidine tag binds to NTA-Ni. The surface modification of the nanoparticles can also be accomplished by utilizing methods such as maleimide chemical conjugation and chemical conjugation of peptide to the nanoparticles surfaces using PEG spacers, or other suitable methods. The preformed nanoparticles are coated as described and incubated for a period of time such that a bond is formed between the 6 histidine and NTA-Ni.

(41) As used herein, the term “liposome” means a type of lipid-based particulate and specifically includes a compartment that is completely enclosed by a lipid bilayer typically composed of phospholipids. Liposomes are prepared according to standard techniques known to those skilled in the art. Emulsion, polymeric, silica, carbon nanotubes, rare metals, silver and/or gold nanoparticle carrier systems may also encapsulate an active agent and be modified on the surface with a drug delivery enhancer for topical applications. Examples of suitable polymeric nanoparticles are PLGA, poly(D,L-lactide-co-glycolide), poly(D,L-lactide), poly(D,L-lactide-co-lactide), poly(L-lactide), poly(glycolide), poly (L-lactide-co-glycolide), poly(caprolactone), poly(glycolide-co-trimethylene carbonate), poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(4-hydroxybutyrate), poly(ester amide), poly(ester-sulfoester amide), poly(orthoester), poly(anhydride), and polysaccharides, such as alginate and chitosan.

(42) The active biomedical or pharmaceutical agent(s) can include small molecules, proteins or peptides, alone or in combination with other small molecules, proteins and/or peptides. The examples below serve to further illustrate the invention, to provide those of ordinary skill in the art with a complete disclosure and description of how the nanostructures or methods herein are made and evaluated, and are not intended to limit the scope of the invention.

(43) In the following examples, luciferin was encapsulated within a lipid nanocarrier system (“Nano-Luc”) for continuous prolonged sustained delivery once administered through variable routs such as IV, IP, and subcutaneous (SQ) delivery. Lipid nanoparticles have been shown to protect the active ingredients from enzymatic degradation, provide controlled release of the active drug, and enhance the therapeutic effect and stabilization of chemically unstable drugs due to lipid matrix (26-28). Along with the Nano-Luc, nanoparticles were developed containing XenoLight DiR (near infrared region fluorescence dye) and luciferin for multimodality imaging of tumors. These were known as NanoLuc-DiR.

Example 1

(44) Lung cancer is one of the leading causes of deaths (1.3 million deaths annually) worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Vascular endothelial growth factor (VEGF) over-expression (61% to 92% of NSCLC) is associated with poor survival. Recently, new approaches in the treatment of lung cancer with novel drugs, which selectively inhibit tumor blood supply, thus controlling cancer cell survival, proliferation and/or metastasis, in combination with conventional anticancer or antiangiogenic drugs, have generated clinical interest. DIM-C-pPhC6H5 (DIM-P), a c-substituted diindolylmethanes is a recent anti-cancer agent. Objectives of this study were: (1) to formulate tumor homing pegylated CREKA peptide coated nanoparticles of DIM-P (PCNCs-D)/D-luciferin (PCNCs-Dl)/XenoLight-DiR (PCNCs-Di); and (2) to evaluate in vivo imaging of tumor progression/tumor vasculature and tracking of nanoparticle delivery system.

(45) Nanoparticles were prepared with DIM-P (NCs-D)/D-luciferin (NCs-Dl)/XenoLight-DiR (NCs-Di), Compritol, Miglyol, DOGS-NTA-Ni and sodium taurocholate using a high pressure homogenizer (Nano-DeBEE). PCNCs-D and PCNCs-Dl/PCNCs-Di were prepared by conjugating NCs-D and NCs-Dl/NCs-Di with 6His-PEG2K-CREKA peptide and characterized for physical properties, clot binding assay and tube formation assay.

(46) Pharmacokinetic parameters of formulations were evaluated in BALB/c mice. In vivo imaging of tumor and tracking of nanoparticles was carried out with IVIS® Spectrum CT (Caliper Life Sciences) by using fluorescent dye (XenoLight DIR) and bioluminescence (luciferin) following intravenous and inhalation delivery of nanocarriers. In vivo imaging following exposure of PCNCs-DL/PCNCs-Di demonstrated their targeting to the tumor vasculature (see FIG. 2), where the PCNCs-Di were found to migrate more in newly formed blood vessels, and total radiant efficiency [p/s]/[μW/cm.sup.2] was 2.1*10.sup.12±0.5*10.sup.12 over the period of 0.5 h to 3 h. NCs-Di did not show any specific migration to tumors confirming the specific targeting of PCNCs-Di, and total radiant efficiency [p/s]/[μW/cm.sup.2] was 0.6*10.sup.12±0.18*10.sup.12.

Example 2

(47) The objective of this study was to formulate nanoparticles of D-luciferin (Nano-Luc), XenoLight-DiR (Nano-DiR) and dual probe nanoparticles with DiR and Luciferin (NanoLuc-DiR) for enhanced in vivo imaging of tumor progression, tumor vasculature and tumor multimodality as well as tracking of the nanoparticle delivery system. Nano-Luc and NanoLuc-DiR were prepared using different lipids for imaging studies. Nanoparticles were characterized for loading and entrapment efficiency, physical properties, release profile, toxicity and stability. Response Surface Methodology (RSM) was utilized to optimize the nanoparticles using design of experiment (DOE Vr.8.0).

(48) Nano-Luc was evaluated against free luciferin for their pharmacokinetic parameters in mice. In vivo imaging of tumors and tracking of nanoparticles was carried out with an IVIS® SpectrumCT (Caliper life Sciences) using a murine xenograft, orthotopic and metastatic tumor models using different cell lines by subcutaneous, intraperitoneal, and intravenous administration of nanoparticles.

(49) Particle size of Nano-Luc and NanoLuc-DiR were found to be <200 nm. Nano-Luc formulation showed a slow and controlled release up to 72 hr (90%). The optimized Nano-Luc had loading efficiency of 5.0 mg/ml with 99% encapsulation efficiency. Nano-Luc and NanoLuc-DiR formulations had good shelf stability, with less than 1% release over one month storage at room temperature and 10% release with accelerated stability testing at 40° C. Pharmacokinetic parameters showed that compared to quick in-and-out of free luciferin, Nano-Luc and NanoLuc-DiR enhanced plasma half-life of luciferin by longer circulation in plasma for more than 24 hr. Tumor multimodality was detected using spectrum CT/DLIT/FLIT imaging of subcutaneous tumor model in mice followed by NanoLuc-DiR administration. Nano-Luc and NanoLuc-DiR were seen to provide enhanced in vivo imaging for tumor diagnostic/detection and multimodality of tumors.

(50) The efficiency and stability of Nano-Luc and NanoLuc-DiR were evaluated in nu/nu and Balb/c mouse models injected with tumor cells (lung and breast tumor cells) expressing the luciferase reporter gene (16, 29-30). The formulation for drug loading, entrapment efficiency and release of luciferin were characterized. Factors that affect these parameters, such as lipid formulation, ratio of lipids/oil/surfactant and process variables, were investigated and optimized by quality by design approach. The formulations were also characterized for stability by accelerated stability studies and differential scanning calorimetry.

(51) Materials

(52) Luciferin and Xenolight DiR were obtained from Calipers-Life Sciences & Technology—A Perkinelmer Company (Alameda, Calif.). The triglyceride Miglyol 812 was obtained from Sasol Germany GmbH (Witten, Germany), and other lipids were obtained from Gattefosse (Saint Priest, France). Dialysis tubing (Molecular weight cut off 6000-8000 Daltons and flat width of 23 mm) was obtained from Fisher Scientific (Pittsburgh, Pa.). Polyoxyethylene-20 oleyl ether or Volpo-20 (Oleth-20) was obtained from Croda Inc (New Jersey, USA). Vivaspin centrifuge filters (Molecular weight Cut off: 10,000 Daltons) were procured from Sartorius Ltd, (Stonehouse, UK). Fetal bovine serum (FBS) and antibiotics were procured from Invitrogen Corp (Eugene, Oreg.). The lung cancer cell lines (A549-luc, H460-luc) and breast cancer cell lines (4T1-luc, MDA-MB-231-luc) were obtained from Perkinelmer Company (Alameda, Calif.). The cells were maintained with supplemented media at 37° C. in the presence of 5% CO.sub.2 in air. All other chemicals used in this study were of analytical grade.

(53) Animals

(54) Male Balb/c mice (20-25 g; Charles River Laboratories) were utilized for the studies. The protocol for in vivo experiments was approved by the Animal Care and Use Committee, Caliper Life Sciences—A Perkinelmer Company, Alameda Calif. The animals were acclimated to laboratory conditions for one week prior to experiments and were on standard animal chow and water ad libitum. The temperature of room was maintained at 22±1° C. and the relative humidity of the experimentation room was found in the range of 35-50%.

(55) Preparation of Nano-Luc and NanoLuc-DiR

(56) Nano-Luc and NanoLuc-DiR were prepared by hot melt homogenization (31). Luciferin and/or Xenolight DiR were dissolved in organic solvent and mixed with lipid phase comprised of different lipids. Organic phase was removed by rota-evaporator for 30 min at 60° C. The lipid phase was mixed with the aqueous phase (20 mL) containing surfactant at the same temperature using a Cyclone IQ2 with Sentry™ Microprocessor (USA) at 20,000 rpm for five (5) min. This mixture was passed through Nano-DeBee® (BEE International, South Easton, Mass.) at 20,000-30,000 psi for three to five cycles. Throughout the process, temperature was maintained at 60° C.

(57) Response Surface Methodology (RSM)

(58) A response surface design was used to evaluate how responses behave at all the studied variables in the experimental region using quadratic polynomial equation. The aim of RSM is to determine the conditions that provide process and product improvement (32). The objective of the present study was to select the lipid, oil and surfactant for the Nano-Luc formulation with the desired response. The particle size, entrapment efficiency, loading efficiency and release rate at 24 hr were used as dependent variables. The actual values of independent variables are reported in Table 1. The parameter level selection was based on a preliminary study and on findings in the literature (Table 1). Design-Expert software (V. 8.0.7.1, Stat-Ease Inc., Minneapolis, Minn., USA) was used for the generation and evaluation of the statistical experimental design.

(59) TABLE-US-00001 TABLE 1 Variables in response surface design. Levels 1 2 3 Independent Variables A: Luciferin (mg) 50 100 200 B: Lipids (700 mg) Monosteol Precirol Geleol C: Oils (330 mg) Miglyol MCT oil Transcutol D: Surfactant (480 μL) Tween 20 Tween 80 Mixture (1:1) Dependent Variables Y1: Mean Particle Size (nm) Y2: Entrapment Efficiency (%) Y3: Loading Efficiency (%) Y4: 24 h Release rate (%)

(60) Central Composite Design

(61) A central composite design was utilized to further optimize significant factors and to assess main, interaction and quadratic effects of the factors on properties of Nano-Luc. Lipid and oil concentrations were selected as significant factors based on the RSM optimization desirability study results. Each of the factors was tested at five (5) different levels and five (5) center points were included. Design-Expert software was used for the design, analysis and plotting of the various 3D and contour graphs.

(62) Optimization of Responses Using Desirability Function

(63) The multiple response method makes use of an objective function called the desirability function. It reflects the desirable ranges for each response (di). The desirability for each response can be calculated at a given point in the experimental domain. The optimum is the point with the highest value for the desirability. The entrapment efficiency and loading efficiency were targeted to maximum, while particle size and release rate were limited to <200 nm and <50%, respectively, in the procedure, as these values confirm the desired product outcome. The desirability function of these parameters was calculated using Design-Expert software.

(64) Characterization of Nano-Luc and NanoLuc-DiR

(65) The particle size and zeta potential of nanoparticles were measured using Nicomp 380 ZLS (Particle Sizing Systems, Port Richey, Fla.). To measure the total amount of drug present in the system, 0.1 ml of formulation was mixed with 0.9 ml of tetrahydrofuran, and the mixture was centrifuged at 5000 rpm for 5 min. The supernatant was collected and absorption was measured at 327 nm.

(66) Entrapment efficiency was determined using vivaspin centrifuge filters. 0.5 ml of formulation was placed on top of the vivaspin centrifuge filter membrane (molecular weight cut-off was 10,000 Daltons) and centrifuged for 20 min at 5000 rpm. About 20 μl flow-through was collected out of 500 μl at the bottom of vivaspin filter, and absorption was measured at 327 nm to determine the luciferin content. The drug loading was determined by centrifuging 1.0 ml of formulation at 16,000 g for 1.5 h, and sediment was dissolved in tetrahydrofuran. The content of luciferin was measured by absorption at 327 nm. Drug loading was calculated using following equation (33):

(67) $\begin{array}{cc}\mathrm{Luciferin}\mathrm{Content}\left(\%\frac{w}{w}\right)=\frac{\mathrm{mass}\mathrm{of}\mathrm{Luciferin}\mathrm{in}\mathrm{nanoparticle}\times 100}{\left(\mathrm{mass}\mathrm{of}\mathrm{nanoparticle}\mathrm{recovered}\right)}& \left(1\right)\end{array}$

(68) Drug Release Studies

(69) Drug release studies were performed using USP 1 (basket) dissolution apparatus (Vankel, N.C.). One (1) ml of nanoparticle formulation was placed in a soaked cellulose membrane (6000-8000 molecular weight cut off), and ends were closed and placed inside the basket. The dissolution media (200 ml) was phosphate buffer saline (PBS) pH 7.4, containing 0.5% w/v Volpo-20 and 0.5% v/v Tween 80. The baskets were rotated at 50 rpm for 72 h at 37.0±0.1° C. The samples (0.5 ml) were collected at different time points with replacement of equal dissolution media. Luciferin content was measured at 327 nm.

(70) Differential Scanning Calorimetry

(71) The interaction of luciferin and Xenolight DiR with lipids and association of the nanoparticle formulation was evaluated using a D SCQ100 (TA instrument, DE). About 5 mg of formulation was weighed and sealed in an aluminum hermetic pan, and the thermal pattern was determined against an empty pan from 0° C. to 300° C. at 5° C. min.sup.−1 heating rate. Transition temperatures were determined from the endothermic peak minima, while transition enthalpies were obtained by integration of the endothermic transitions using linear baselines. Graphical illustrations of observations are shown in FIGS. 8A-8F.

(72) Accelerated Stability Studies

(73) Nano-Luc and NanoLuc-DiR were stored at different temperatures 30±1° C., 40±1° C. and 50±1° C., along with at room temperature protected from light (mean temperature being 25.7±0.6° C.), for a month (34). Aliquots were removed after intervals of time (0 days, 7 days, 14 days, 21 days and one month), and formulations were analyzed for particle size, entrapment efficiency, release rate and luciferin content by methods described previously. As depicted in FIG. 9, a graph was plotted between log % luciferin remaining vs. time. The slope of the curve was determined from the graph, and degradation rate constant (K) was calculated by using the equation:

(74) $\begin{array}{cc}\mathrm{Slope}=\frac{K}{2.303}& \left(2\right)\end{array}$ (2)
Where, K is the degradation rate constant.

(75) The effect of temperature (30° C., 40° C. and 50° C.) on degradation was studied by plotting log K vs. 1/T (Kelvin.sup.−1) (Arrhenius plot), as seen in FIG. 9. Further, the value of K at 25° C. and 8° C. (K.sub.8 and K.sub.25) was extrapolated from the Arrhenius plot, and shelf life at both room temperature (25° C.) and refrigerator temperature (8° C.) was predicted from Eq. (2).

(76) In Vivo Tumor Models

(77) In vivo tumors were grown using lung cancer cell lines (A549 and H460) and breast cancer cell lines (4T1 and MDA-MB-231). All the cell lines were modified for the luciferase reporter gene expression.

(78) Xenograft Tumor Model

(79) The adherent tumor cells were washed with PBS, harvested from conXuent cultures by 5-min exposure to 0.25% trypsin and 0.02% EDTA solution in an incubator. Trypsinization was stopped with medium containing 10% FBS. The cells were centrifuged, and the floating cells in the supernatant were discarded. The cell pellet was resuspended in medium containing 10% FBS and mixed thoroughly. Trypan blue staining was used to determine the number of viable cells. The resuspended cells were centrifuged, and cell dilutions of 2±10.sup.6 cells/100 μl were prepared in growth medium. The 100 μl of cell suspension was injected subcutaneously into the right flank area of each mouse. The mice were randomized into control and treatment groups when xenografts were palpable with a tumor size of 50 mm.sup.3.

(80) Orthotopic Tumor Model

(81) The Orthotopic Tumor Model was Used to Mimic the Cancer in Humans in Athymic nu/nu mice (6-week old). Mice were anesthetized and a 5 mm skin incision was made to the left chest, 5 mm below the scapula. Hamilton syringes (1 mL) with 28-gauge hypodermic needles were used to inject the cell inoculums through the sixth intercostal space into the left lung. The needle was advanced to a depth of 3 mm and removed after the injection of the cells (1×10.sup.6 per mouse) suspended in 100 μLPBS (pH 7.4) into the lung parenchyma. Only cell suspensions of >90% viability, as determined by trypan blue exclusion, were used. Wounds from the incisions were closed with surgical skin clips. Animals were observed for 45 to 60 min until fully recovered. Mice developed lung tumors 10-14 days after inoculation of the cells, and mice were randomized in various groups after 10 days post tumor implantation.

(82) Metastatic Tumor Model

(83) Nu/Nu mice were anesthetized and tumor cells (2 million per mouse) were injected via tail vein. Only cell suspensions of >90% viability, as determined by trypan blue exclusion, were used. A pilot study showed that all the nude mice develop lung tumors at 10-14 days after intravenous injection of tumor cells. Ten days after tumor implantation, the animals were randomly divided into groups to receive treatment.

(84) In Vivo Imaging

(85) Bioluminescence/Fluorescence Imaging

(86) Mice were anesthetized with isoflurane and imaged for different time points up to 24 hr following IP, IV and SQ injection of 150 mg/kg luciferin solution, Nano-Luc (equivalent to 150 mg/kg of luciferin) and NanoLuc-DiR (equivalent to 150 mg/kg of luciferin). Imaging was performed with an IVIS Spectrum (16). Bioluminescent signals were quantified using Living Image® software (Caliper Life Sciences., Alameda, Calif.).

(87) Tumor Multimodality (CT/DLIT/FLIT) Imaging

(88) Mice were anesthetized with isoflurane and imaged following IP, IV and SQ injection of NanoLuc-DiR (equivalent to 150 mg/kg of luciferin). Imaging was performed with an IVIS SpectrumCT (35). Bioluminescent signals were quantified using Living Image® software (Caliper Life Sciences., Alameda, Calif.).

(89) Statistical Analysis

(90) Pooled data were expressed as mean±standard deviations (SD) and model parameters as estimates±standard errors (SE). Means were compared between two groups by t-test and between three dose groups by one-way variance analysis (ANOVA); data were explored for two-way ANOVA analyses, where applicable. Correlations between doses and parameters were sought by use of the linear regression coefficient (r) and the coefficient of determination (R.sup.2). Probability (p) values<0.05 were considered significant. All statistical analyses were performed using GraphPad Prism® 5.0 software (San Diego, Calif.).

(91) Results

(92) Experimental Design and Effect of Variables on Response

(93) Luciferin containing nanoparticles were prepared using a hot melt homogenization method. The experimental runs with variables and corresponding responses for the 32 formulations tested are presented in Table 2.

(94) TABLE-US-00002 TABLE 2 Presentation of experiments with actual values for factor levels in design with their responses for particle size, entrapment efficiency, loading efficiency and 24 hr release rate. Each experiment was performed using three (3) replicates of different nanoparticles. Y1: Mean Y2: Entrapment Y3: Loading Y4: 24 hr A: Luciferin B: Lipids C: Oils D: Surfactant Particle Size Efficiency Efficiency Release Rate Run mg mg mg ul nm % % % 1 100 Monosteol Miglyol Mixture of Tween 20 and Tween 80 179 99 89 50.89 2 50 Geleol MCT oil Tween 20 168 97 75 42.68 3 50 Presirol MCT oil Tween 80 180 94 76 59.64 4 200 Geleol MCT oil Mixture of Tween 20 and Tween 80 173 95 69 39.89 5 100 Presirol Transcutol Tween 80 168 94 60 59.32 6 100 Presirol Miglyol Tween 80 179 96 90 49.98 7 50 Geleol Miglyol Mixture of Tween 20 and Tween 80 200 97 91 57.5 8 200 Geleol Transcutol Tween 80 210 95 67 48.36 9 100 Monosteol MCT oil Tween 20 179 96 82 45.65 10 50 Monosteol MCT oil Mixture of Tween 20 and Tween 80 168 91 86 50.25 11 200 Presirol Miglyol Mixture of Tween 20 and Tween 80 187 93 91 57.32 12 100 Presirol Transcutol Tween 80 188 92 67 54.32 13 100 Presirol Miglyol Tween 80 175 95 92 59.35 14 200 Presirol Transcutol Mixture of Tween 20 and Tween 80 172 89 59 61.32 15 200 Presirol MCT oil Tween 80 198 82 81 56.31 16 200 Monosteol MCT oil Mixture of Tween 20 and Tween 80 189 92 84 48.36 17 200 Monosteol Miglyol Tween 20 192 96 91 47.69 18 50 Presirol Transcutol Mixture of Tween 20 and Tween 80 167 89 65 44.36 19 100 Presirol MCT oil Mixture of Tween 20 and Tween 80 158 89 72 48.36 20 100 Monosteol MCT oil Tween 20 167 97 81 47.68 21 50 Monosteol Transcutol Tween 20 162 94 73 44.98 22 200 Presirol MCT oil Tween 20 176 86 64 56.35 23 50 Monosteol Miglyol Tween 80 164 97 81 48.55 24 200 Monosteol Transcutol Tween 80 167 95 69 43.98 25 200 Geleol Miglyol Tween 80 173 91 84 47.85 26 100 Monosteol Miglyol Mixture of Tween 20 and Tween 80 149 92 88 49.35 27 100 Geleol Transcutol Mixture of Tween 20 and Tween 80 158 87 56 48.65 28 50 Presirol Transcutol Tween 20 164 88 57 32.89 29 200 Geleol Transcutol Tween 20 187 91 68 68.35 30 100 Geleol Miglyol Tween 20 188 92 87 61.34 31 100 Geleol Miglyol Tween 20 172 89 91 59.39 32 100 Geleol MCT oil Tween 80 195 82 78 54.85

(95) As can be seen in Table 2, the mean particle size ranged from 149 nm to 210 nm depending on the factor level selected during preparation. The response surface quadratic model was used for analysis purposes. Statistical analysis revealed that none of the factors were significant to influence mean particle size (Y1), as shown in Table 3.

(96) TABLE-US-00003 TABLE 3 This table illustrates statistical analysis of mean particle size (Y1), entrapment efficiency (Y2), loading efficiency (Y3), and 24 hr release rate (Y4) in the Response Surface design. Y1: Mean Particle Y2: Entrapment Y3: Loading Y4: 24 hr Size Efficiency Efficiency Release Rate F value p value F value p value F value p value F value p value A: Luciferin 2.4297 0.1798 1.3226 0.3021 0.1213 0.7418 6.3462 0.0453* B: Lipids 1.0120 0.4275 2.7413 0.1571 10.0924 0.0176* 6.9900 0.0271* C: Oils 0.0677 0.9353 6.3760 0.0421 149.5454 <0.0001* 2.3294 0.1784 D: Surfactant 2.1018 0.2175 1.1472 0.3890 0.8709 0.4736 0.6808 0.5414 *Significant values at p < 0.05

(97) The entrapment efficiency was represented in percentage of loading efficiency, ranging from 82% to 99% depending on the factor level selected during preparation (see Table 2). The response surface quadratic model with inverse transform was used for analysis purposes. Statistical analysis revealed that oils were the significant factor to influence entrapment efficiency (Y2), as seen in Table 3. As shown in Table 2, for all formulations the loading efficiency (Y3) of Nano-Luc was in range of 56% to 92%. The most significant factor affecting the loading efficiency was shown to be oils (p<0.05) followed by lipids (p<0.05) used in the preparation of Nano-Luc.

(98) An increase in release rate was observed with increase in luciferin concentration, and lipids were also significantly influencing the release rate. Effect of lipid- and oil-type factors on loading efficiency, entrapment efficiency, 24 hr release rate and mean particle size are shown in FIGS. 5A-5D.

(99) Central Composite Design

(100) After the lipids and oils were found as critical factors based on the screening design, a 2-factor, 5-level central composite design was applied to explore the optimum levels of these factors, as seen in Table 4.

(101) TABLE-US-00004 TABLE 4 Presentation of experiments with actual values for variables in central composite design with their responses for entrapment efficiency, loading efficiency, and 24 hr release rate. A-Monosteol B:Migiyol X1: Entrapment X2: Loading X3: 24 hr Run (mg) (mg) Efficiency (%) Efficiency (%) Release rate (%) 1 600 950 86.64 71.58 58.95 2 600 950 85 74.95 57.69 3 300 400 57.36 79.36 67.05 4 600 250 86.58 72.69 56.98 5 600 250 84.36 74.23 55.89 6 900 100 95.36 58.95 35.68 7 600 462.13 87.25 99.25 61.35 8 900 400 98.35 99.51 45.98 9 1024.26 250 99.9 84.36 34.89 10 300 100 56.98 42.36 69.35 11 600 37.86 97.65 31.35 35.24 12 600 250 84.69 78.25 52.35 13 175.73 250 76.38 95.35 67.25

(102) This methodology included 2 groups of design points, including 2-level factorial design points, axial or star points, and center points (36). Two independent factors were studied at 5 different levels, coded as −α, −1, 0, 1, and +α, to determine the main, interaction and quadratic effects of the solute and Soluplus concentrations on the selected responses. The value for alpha (1.414) was intended to fulfill the rotatability in the design. The other variables were fixed at the following values: luciferin (100 mg); surfactant (480 μl). The experimental runs with formulation variables and corresponding responses for the 13 tested formulations are presented in Table 4. The best fit for each of the responses was found for the quadratic models of Y1 and Y2, and the linear model of Y3. The statistical analyses for response following analyses of the models were described as the effect of various factors on the tested responses, as seen in Table 5.

(103) TABLE-US-00005 TABLE 5 Statistical analysis of entrapment efficiency (X1), loading efficiency (X2) and 24 hr release rate (X3) in the Response Surface design. X1: Entrapment X2: Loading X3: 24 hr Efficiency Efficiency Release Rate p p p EC value EC value EC value Intercept 85.454 N/A 74.2 N/A 0.01780 N/A A-Monosteol 14.0790 0.0046 2.6497 0.4148 0.0049 0.0002 B-Miglyol −1.4172 0.6930 21.6981 0.0002 −0.0028 0.0053 AB 0.6525 0.8972 0.89 0.8428 −0.0016 0.1389 A{circumflex over ( )}2 −1.9776 0.6089 5.9443 0.1127 0.0014 0.1094 B{circumflex over ( )}2 0.1773 0.9630 −6.3331 0.0947 0.0016 0.0632 EC (Estimated Coefficient) *Significant value at p < 0.05

(104) Contour plots and three-dimensional response surfaces were drawn to estimate the effects of the independent variables on each response (FIGS. 6A and 6B). The overall desirability response was calculated from the individual desirability of each of the responses using DOE v8.0.7. The optimized composition was identified with a desirability value of 0.968 (FIGS. 7A and 7B).

(105) Differential Scanning Calorimetry (FIGS. 8A-8F)

(106) For the free luciferin, the thermogram revealed a small, clear event at about 197° C. (FIG. 8A). No melting was observed before or during the process which can therefore most likely be attributed to a solid-solid phase transformation. The new modification was stable upon cooling. Upon further heating, a very pronounced DSC endothermic peak appeared at about 237° C. (FIG. 8A), and subsequently, the drug decomposed and turned black. The DSC thermogram of precirol and monosteol alone showed a sharp endothermic peak at about 63° C.

(107) Following the addition of miglyol and luciferin, there was a depression in the endothermic peak mainly because these entities behave as impurities. Also, DSC studies were performed to confirm the absence of drug excipients interactions. The DSC thermograms of physical mixture of components are shown in FIG. 9 in the same ratio as formulation and Nano-Luc. The DSC thermogram showed a sharp endothermic peak for luciferin at 197° C. and for precirol at 63° C. No considerable shift in the position of endothermic peaks was observed in the DSC thermogram of physical mixture.

(108) Stability Studies

(109) Accelerated stability studies were conducted on Nano-Luc using the particle size, loading efficiency, and entrapment efficiency as the prime parameters. There was a slight increase in the particle size during the one-month storage from the 172±5.62 nm to 188.56±7 0.80 nm with not much change in PDI (i.e., initially it was 0.330±0.06 and after 1 month it was 0.348±0.01).

(110) The entrapment efficiency and loading efficiency (%) of Nano-Luc batch initially was found to be 97.66±2.72% and 96.12±3.86%, respectively. After a month, the entrapment efficiency and loading efficiency of Nano-Luc batch was found to be 96.67±0.14% and 94.12±2.34%, respectively, indicating that the drug can be retained within the nanoparticles for the sufficient period of time. Also, the accelerated stability studies at 30° C., 40° C. and 50° C. were conducted and percentage recovery of luciferin from Nano-Luc was measured at different time points, as seen in FIG. 9.

(111) Regarding storage of the Nano-Luc, there were no significant alterations in the size, PDI and entrapment efficiency of the nanoparticles. There was a decrease in loading efficiency at 50° C., since the melting point confirmed by DSC (FIGS. 9B and 9D) was near 60° C. A possible reason is the alteration of the lipid matrix and leaching of luciferin from the caged matrix.

(112) In Vivo Imaging and Kinetics of Free Luciferin and Nano-Luc/NanoLuc-DiR

(113) Free luciferin was cleared from circulation within 60 min, as is apparent in 5 min and 120 min images in 4T1-luc models. With a matched intensity color map and total injected luciferin, the images acquired for Nano-Luc luciferin in formulations are compared to that of free luciferin for the corresponding animal models (FIGS. 12A-12C). General trends of short circulation of free luciferin, fast release from Nano-Luc (IV), and slow release from Nano-Luc (IP/SC) over time were observed for all tumor models.

(114) Bioluminescence intensities quantified by drawing an ROI on each tumor as well as on the lower back of the animal showed similar PK profiles. The bioluminescence images were then quantified and used to evaluate the pharmacokinetics of free and Nano-Luc luciferin in 4T1-luc tumor models, as seen in FIGS. 10A and 10B). A rapid clearance of free luciferin was observed with a similar kinetic in all models (FIG. 12A-12B) with an estimated t.sub.1/2 value of more than 2 hr in IP/SC delivery of Nano-Luc. Intravenous Nano-Luc luciferin, however, showed a two phase release kinetic, a rapid release in the early phase (t<30 min), followed by a slower steady release kinetic (FIG. 12C).

(115) As seen in FIG. 12C, radiance resulting from the injection of Nano-Luc increased over the first hour and remained steady for another 4 hr and started declining slowly. However, radiance was detectable over 24 hr. With free luciferin, during the first 30 minutes, a rapid increased in radiance was observed; after 30 minutes, a rapid decline can be seen. Luciferin loaded in Nano-Luc had a 400-fold greater phase II half-life in circulation as compared to free luciferin.

(116) The multimodality (Spectrum CT/FLIT/DLIT) imaging of 4T1-luc tumor model was visualized using 3D construct of tumor and mice, followed by administration of NanoLuc-DiR. A similar trend was observed with respect to luciferin release and expression of bioluminescence intensities from NanoLuc-DiR formulations (FIG. 13A) as compared to Nano-Luc. Additionally, florescence intensities were steady over the period of time (FIG. 13B).

DISCUSSION

(117) Luciferase reporters are established to analyze molecular and cellular events with simple, cost-effective, extremely sensitive and non-invasive method to image biologic processes in vivo (37) using bioluminescence imaging (38). Luciferin (e.g., a firefly luciferin substrate) is an amphipathic molecule with a relatively short half-life of 5.33 min (25) and permeability co-efficient of 3.6×10.sup.−9 cm.Math.s.sup.−1 (25). Due to the faster clearance of luciferin from plasma and limitation of multiple injections of luciferin (25), the foregoing study proposed controlled sustained release of luciferin with effective radiance for imaging over the period of 24 hr.

(118) For the formulation of luciferin as Nano-Luc, monosteol and precirol were found to be the most suitable lipids due to the higher solubility and partitioning. High amounts of mono-, di-, and triglycerides present in lipids help the drug to solubilize in the lipid fraction. Miglyol provides additional space for drug molecules to get entrapped, thus enhancing drug loading (39).

(119) For the optimization of Nano-Luc, response surface experimental design showed a significant correlation between dependent and independent factors. Quadratic model was found to be the most suitable for defining the relationship for all the responses (model F value<0.05; lack of fit value>0.05 as per one-way ANOVA). Central composite design showed correlation between lipid concentrations and response variable entrapment efficiency, loading efficiency and release of luciferin from Nano-Luc.

(120) After the analysis of data, optimization using DOE Vr 8.0.7 software was performed to get a particle size of less than 200 nm with maximum entrapment efficiency and loading efficiency with 50 percent drug release at 24 hr. The intention behind these particular selections was to provide controlled sustained release of luciferin. FIG. 14 depicts the release rate of luciferin from Nano-Luc in in vitro conditions over time.

(121) Using these criteria, the three variables were then combined to determine an overall optimum design. FIGS. 5A-5D show an acceptable region that describes the requirements of these responses. This optimum region could therefore be used to construct the design space of Nano-Luc with high quality characteristics. A further central composite design optimization and validation process was then undertaken using desirable characteristics (FIGS. 7A and 7B) that depended on the prescriptive criteria of maximum entrapment efficiency, maximum loading efficiency and release rate.

(122) The DSC thermograms of monosteol, miglyol, precirol and Nano-Luc were represented in FIGS. 8A-8F. The DSC study concluded with the absence of any chemical interaction between luciferin and excipient. The luciferin endothermic peak was not observed in the DSC thermogram of Nano-Luc, likely at least in part because of the molecular inclusion of luciferin in the lipid matrix.

(123) Similar results have been reported earlier by other researchers (40-41). Puglia et al. (42) observed that the addition of ketoprofen or naproxen to the lipid formulations resulted in the broadening of the lipid endothermic peak. DSC thermograms of Nano-Luc showed broadening of lipid peak, and the reasons for this observation may be (1) the excipients undergoing several heating and cooling cycles, (2) the smaller size of the particles contributing to a larger surface area, and (3) miglyol, luciferin and surfactant behaving as impurities.

(124) It was found that entrapment efficiency of nanoparticulate formulation was not changed drastically during stability studies, indicating that the formulation was stable at specified storage condition up to one month. The stability data indicated that the lipids have contributed to the stabilization of the formulation and could be useful for improving the shelf life of Nano-Luc. This might be attributed to the fact that transformation of colloidal suspension into solid form has the advantages of preventing particle aggregation, degradation reactions (hydrolysis), and leakage of the drug. Furthermore, the shelf-life of Nano-Luc estimated at 25° C. was more than ten months, while at 8° C. it was more than two years.

(125) To evaluate the in vivo effectiveness of Nano-Luc, nanoparticles were administrated via SC, IP and IV route into mice having tumor expressing luciferase. Free luciferin cleared rapidly (within 60 min) with a biphasic time course. Luciferin encapsulated in Nano-Luc remained in the system giving radiance enough for imaging for more than 24 hr. For a period of 20 min, Nano-Luc formulations demonstrated an early and rapid release similar to free luciferin by IV administration. On the contrary, SC and IP administration of Nano-Luc demonstrated slow release of luciferin in vivo compared to free luciferin, with peak intensity lower than that of free luciferin. This phenomenon was also observed by Gross et al (21) for osmotic pump delivery of luciferin.

(126) Table 6 illustrates the significant differences between certain embodiments of the current invention and the research disclosed in Kheirolomoom et al. (25) and Gross et al. (21).

(127) TABLE-US-00006 TABLE 6 Comparison of the current methodology with the conventional methodologies of Kheirolomoom et al. (25) and Gross et al. (21). Luciferin liposome Luciferin Osmotic pump Nano-Luc (Kheirolomoom et al.) (Gross et al.) Method of preparation Hot-melt Thin lipid film hydration Micro-osmotic pumps (Alzet Model homogenization followed by extrusion 1007D, 0.5 μL/h release rate, 100 μL, at high speed reservoir; Durect, Cupertino, CA) followed by were loaded with d-luciferin (50 few cycles of mg/mL in sterile phosphate-buffered high pressure saline [PBS]) under aseptic homogenization conditions according to the manufacturer's instructions Delivery method Intravenous, Intra-tumoral, intravenous Surgically implanted intra-peritoneal, (subcutaneously) in the dorsal neck subcutaneous fat pad Components Luciferin, solid Luciferin, pH buffers, Luciferin and sterile phosphate- lipid, oil (liquid solid lipid, polyethylene buffered saline lipid) e.g. glycol, e.g., DPPC, lyso- miglyol, palmitoyl PC, DSPE- monosteol, PEG2k, SoyPC, precirol, etc. cholesterol Luciferin loading type/ Passive Passive loading: max 28.8 100 μL of 50 mg/mL Luciferin: efficiency loading: 2-10 ug/mg of lipid mg/ml; or Active loading: max 172 50-250 ug/mg ug/mg of lipid of lipid Encapsulation/Entrapment >98% 90-95% N/A efficiency of luciferin

(128) Apart from these differences and the advantages of the current invention that are readily apparent, bioluminescence kinetics was steady over the period of 4 hr utilizing embodiments of the current invention at peak flux, while the kinetic profile of the liposomes shows no steady flux but steady decrease in luciferin kinetics (Kheirolomoom et al. (25)). Additionally, as described previously, imaging with embodiments of the current invention was possible over 24 hr, while with liposomes in Kheirolomoom et al. (25), imaging was possible over 12 hr.

(129) The osmotic pump delivery disclosed in Gross et al. (21) showed release and imaging possible for 48 hr; however, raw bioluminescence compared to free luciferin was 70-150 folds lower with the osmotic pump. Moreover, the bioluminescence signal was not steady in photon count, while with embodiments of the current invention provide a steady bioluminescence signal for 4 hr.

(130) Regarding preparation for administration, Kheirolomoom et al. (25) requires local hyperthermia to be induced with ultrasound to heat the tumor area at 42° C., in order to increase the raw bioluminescence radiance. Overall, in Kheirolomoom et al. (25), without hyperthermia, radiance was about 75 times lower relative to free luciferin; with hyperthermia, radiance was still about 25 times lower relative to free luciferin. It was found that radiance was only about 10 times lower relative to free luciferin when utilizing the current methodology. The liposome's raw bioluminescence with hyperthermia in Kheirolomoom et al. (25) at equivalent molar concentration of luciferin was about 30-50% lower than that of Nano-Luc without using any local hyperthermia.

(131) With IV administration, flux efficiency of Nano-Luc luciferin was higher than that of free luciferin, which may be due to faster clearance of free luciferin from the system than Nano-Luc. This rapid release of luciferin was followed by a slow release in the second phase, which was more likely the release of the encapsulated luciferin. The early rapid release observed for luciferin in Nano-Luc likely results from the total luciferin that was initially associated with the outer core of Nano-Luc (39).

(132) Whereas the osmotic pump delivery approach by Gross et al (21) requires surgical implantation of device, the current methodology can be performed by simple injection via SC, IP, or IV route.

(133) As per Kheirolomoom et al (25), intravenously injected long circulating luciferin liposomes provided sufficient radiance for more than 12 hr of imaging, while Nano-Luc provided sufficient radiance for 24 hr. Also, in vivo kinetics for the liposome showed declining phase from time of injection as per Kheirolomoom et al (25) and never showed steady phase in luciferin kinetics, further illustrating the low level of raw bioluminescence radiance. In contrast, Nano-Luc showed steady kinetics for about 2 hr and started slowly declining over the remaining period, as seen in FIG. 12C.

(134) Additionally, the multimodal imaging approach using NanoLuc-DiR—including bioluminescence radiance, fluorescence intensity and spectral imaging—has permitted evaluation of the imaging of tumor modality using IVIS® spectrum CT/FLIT/DLIT. The application of this approach can be varied with different parameters. For example, an alternative method permits assessments of targeted therapeutic efficacy while monitoring tumor regression during treatment in vivo. This is in contrast to conventional tumor measurements at the termination of treatment periods. Overall, embodiments of the current invention can significantly shorten the time required for assessing preclinical efficacy.

(135) Further, it enables screening of drug/nanoparticle localization in tumors in vivo with high resolution, quantitatively and specifically. This may be a useful approach to screen a panel of new nano-therapeutics in vivo in order to select an effective nano-therapeutic for further testing of therapeutic efficacy. The characterization of nano-therapeutic particles in vivo would involve attachment of fluorophores to particles to visualize the localization in vivo. Whether the molecules of interest are inherently fluorescent (Xenolight DiR) or labeled with a fluorophore, the multimodality imaging method described herein can provide a powerful approach for characterizing nanoparticle activities in vivo in preclinical studies.

(136) The foregoing study exemplified the development and evaluation of nanoparticles of an in vivo imaging agent luciferin. The prepared Nano-Luc was optimized for its formulation and in vitro parameters. Accelerated stability assessment of prepared Nano-Luc shows the potential of the nanoparticle in protection of the entrapped drug. The calculated shelf life of Nano-Luc was found to be more than 10 months at 25° C. Nano-Luc delivered luciferin over a relatively lengthy period of time, expressing sufficient bioluminescence radiance (within tumors) for more than 24 hours of imaging when administered by IP, SC, or IV to mice expressing luciferase. Nano-Luc kinetic studies revealed a steady and longer release of luciferin when encapsulated as compared to free luciferin. Furthermore, NanoLuc-DiR showed possibility of tumor multimodality imaging, as well as its use for characterizing nanoparticle activities in vivo in preclinical studies.

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Definitions of Claim Terms

(138) Bio-imaging agent: This term is used herein to refer to a molecule that emits a contrast signal, such as the emission of light or color, when catalyzed or on its own. Emission of this contrast signal allows the site, where the bio-imaging agent has been catalyzed, to be imaged in vivo. Various types of bio-imaging agents are contemplated, for example fluorescent dyes, bioluminescent agents, chemi-luminescent agents, carbon nanotubes, metal nanoparticles/nanotubes (e.g., gold, silver, rare metals, selenium, etc.), non-metallics (e.g., silica, porous silica, etc.), gas (e.g., perfluorocarbon, nitrogen, etc.), radio-isotopes, phosphate-based compounds (e.g., disodium etidronate, tin pyrophosphate, polyphosphate4 and sodium trimetaphosphate, etc.), among others.

(139) Effective, detectable biofluorescence: This term is used herein to refer to a contrast signal emitted from the target, wherein the signal has a strength that can be detected using known imaging techniques, for example radiography, MM, nuclear medicine, photo-acoustic imaging, tomography, and ultrasound, among others.

(140) Enhanced half-life: This term is used herein to refer to an increase in the amount of time required for the concentration of a particular reactant to fall from a specific value to half of that specific value. An increased half-life of a reactant would allow that reactant to have a certain effect over a greater period of time. For example, if the in vivo half-life of luciferin is enhanced or increased, one would be able to conduct in vivo bio-imaging for a greater period of time.

(141) Entrapment efficiency: This term is used herein to refer to the ratio of the amount of an active agent (drug) actually encapsulated within a carrier to the amount of that active agent (drug) that was added. A higher entrapment efficiency would typically be desired, as that would indicate a high percentage of active agent added became encapsulated within the carrier, as desired.

(142) Ingredient: This term is used herein to refer to any substance used to formulate and fabricate a nanoparticle carrier as described herein. Examples include, but are not limited to, lipids, metals, polymers, and carbon nanotubes, alone or in combination.

(143) Loading efficiency: This term is used herein to refer to the ratio of the amount of an active agent (drug) in a carrier system to the total weight of that carrier system. A higher loading efficiency would typically be desired, so long as the overall system was effective in its purpose.

(144) Optimized: This term is used herein to refer to an entity, or an aspect thereof, having the most favorable or desirable qualities. There can be a cause and effect relationship between two aspects of an entity, such that optimizing one aspect would cause the other aspect to have the most favorable or desirable qualities. For example, the ratio of ingredients in a nanoparticle carrier should be optimized to allow for maximum entrapment and loading efficiencies, along with a desired release rate. The optimal ratio of ingredients would depend, in part, on the type of ingredients used and the effects desired in the resulting nanoparticle carrier.

(145) Predetermined ratio: This term is used herein to refer to a proportion of the amount of solid phase ingredients used to fabricate a nanoparticle carrier to the amount of liquid phase ingredients used to fabricate that nanoparticle carrier. This ratio is important in that it determines characteristics of the resulting nanoparticle carrier, for example the nanoparticle carrier's entrapment efficiency, loading efficiency, and release rate of the bio-imaging agent or other active substance.

(146) Release rate: This term is used herein to refer to the rate at which an active agent (drug) is released from within its corresponding carrier. Once released, that active agent (drug) can have its intended effects on its target. A desired release rate would depend on the ultimate goal. For example, a drug may be desired to be released all at once after a certain period of time. Alternatively, a drug may be desired to be released constantly and consistently throughout a certain period of time. With bio-imaging, typically one would want the agent to be released constantly throughout the imaging period, so that the target can be imaged throughout the imaging period.

(147) Stable: This term is used herein to refer to the thermodynamic characteristic of a substance or entity being consistent or difficult to modify chemically. Thus, if solid phase and liquid phase ingredients have a predetermined ratio that is stable, then the resulting mixture of the ingredients (e.g., a nanoparticle carrier) would be difficult to modify chemically (e.g., melting, freezing, inactivating, destabilizing, etc.).

(148) Subject: This term is used herein to refer to any animate or inanimate animal body that is submitted to the system and/or method of the current invention, or any aspect thereof. Wide ranges of subjects are contemplated, and examples include, but are not limited to, vertebrate animals, human beings, primates, mice, etc.

(149) Target: This term is used herein to refer to a specific structure that one desires to image and thus should be sufficiently biofluorescent to be imaged. This structure can be natural, such as an internal organ in a human being, or can be artificial, such as tumor cells injected into a mouse.

(150) Target-honing molecule: This term is used herein to refer to a target specifier that enhances delivery of a drug by directing the drug (and the corresponding carrier) to a particular target or goal. This can be accomplished, for example, by the molecules having tags with only specific binding regions that correlate to the target cell type of interest. An example of a target-honing molecule is the CREKA peptide, which targets tumor cells in the body. Thus, this type of target-honing molecule can be called a tumor-honing molecule. Various classes of target-honing molecules are contemplated, for example peptides, proteins, RNA, DNA, SiRNA, etc.

(151) The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained. Since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

(152) It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention that, as a matter of language, might be said to fall therebetween.