MICROPARTICLE COMPOSITIONS

Abstract

There is provided a microparticle composition suitable for molecular imaging, the composition comprising microparticles, wherein the microparticles comprise: a core microparticle structure having a central area and a shell, and wherein the core microparticle structure comprises (i) a phosphatidylcholine lipid: (ii) a phosphatidylethanolamine lipid comprising at least one maleimide moiety; and (iii) an alkoxylated fatty acid.

Claims

1. A method of preparing a microparticle composition suitable for molecular imaging, the microparticle composition comprising microparticles, wherein the microparticles comprise: a core microparticle structure having a central area and a shell, and wherein the core microparticle structure comprises a phosphatidylcholine lipid, a first phosphatidylethanolamine lipid comprising at least one maleimide moiety, and an alkoxylated fatty acid, the method comprising the steps of: a) forming a lyophilisate comprising the phosphatidylcholine lipid, the first phosphatidylethanolamine lipid, and the alkoxylated fatty acid; b) dissolving the lyophilisate in an aqueous-based solution; c) heating the solution to a temperature which exceeds a chain melting temperature (Tc) of the lipids in the lyophilisate; and d) shear-mixing the solution in presence of a gas.

2. The method of claim 1, wherein the temperature in step c) exceeds 60 C.

3. The method of claim 1, wherein the temperature in step c) exceeds 65° C.

4. The method of claim 1, wherein the temperature in step c) is in a range of approximately 60 to 65° C.

5. The method of claim 1, further comprising the step of: e) covalently attaching at least one molecular binding element to the shell of the core microparticle structure.

6. The method of claim 5, wherein the at least one molecular binding element is covalently attached to the core microparticle structure via the at least one maleimide moiety.

7. The method of claim 5, wherein the at least one molecular binding element comprises a protein, peptide, or small organic moiety.

8. The method of claim 7, wherein the at least one molecular binding element is an antibody.

9. The method of claim 5, wherein the conjugation reaction molar ratio of the at least one molecular binding element to the first phosphatidylethanolamine lipid is ≥1:1.

10. The method of claim 9, wherein the conjugation reaction molar ratio of the at least one molecular binding element to the first phosphatidylethanolamine lipid is ≥5:1.

11. The method of claim 5, wherein the microparticles have at least about 1×10.sup.5 molecular binding elements per microparticle.

12. The method of claim 1, wherein the core microparticle structure comprises: (i) a C18-24 saturated phosphatidylcholine lipid in a molar ratio of 72 to 78; (ii) a first C18-24 saturated phosphatidylethanolamine lipid comprising at least one maleimide moiety in a molar ratio of 7 to 12 and a polyethylene glycol chain with a molecular weight of at least 500; and (iii) an alkoxylated fatty acid in a molar ratio of 12 to 18, the alkoxylated fatty acid being a C18-24 saturated polyethylene glycol fatty acid ester.

13. The method of claim 12, wherein the polyethylene glycol fatty acid ester is a PEG40 stearate.

14. The method of claim 12, wherein the at least one maleimide moiety is attached to the polyethylene glycol chain.

15. The method of claim 14, wherein the first phosphatidylethanolamine lipid comprising at least one maleimide moiety is a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000].

16. The method of claim 1, wherein the core microparticle structure is micellar.

17. The method of claim 1, further comprising the step of: e) incorporating at least one labelling moiety into the microparticle composition.

18. The method of claim 17, wherein the labelling moiety is incorporated in the microparticle composition via attachment to at least one of the phosphatidylcholine lipid, the first phosphatidylethanolamine lipid comprising at least one maleimide moiety, and the alkoxylated fatty acid.

19. The method of claim 17, wherein the molar ratio of the labelling moiety in the composition is 0.2 to 50.

20. The method of claim 17, wherein the molar ratio of the labelling moiety in the composition is 0.5 to 5.

21. The method of claim 17, wherein the labelling moiety is a fluorescent dye.

22. The method of claim 21, wherein the labelling moiety is 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI).

23. The method of claim 1, wherein the core microparticle structure further comprises a second phosphatidylethanolamine lipid.

24. The method of claim 23, wherein the second phosphatidylethanolamine lipid is a C.sub.18-24 saturated phosphatidylethanolamine lipid.

25. The method of claim 23, wherein the second phosphatidylethanolamine lipid is a distearoylphosphatidylethanolamine (DSPE).

26. The method of claim 23, wherein a molar ratio of the second phosphatidylethanolamine lipid in the composition is 0.5 to 5.

27. The method of claim 1, wherein the central area of the core microparticle structure contains a fluid medium.

28. The method of claim 27, wherein the fluid medium comprises a physiologically acceptable gas.

29. The method of claim 28, wherein the physiologically acceptable gas is selected from air, nitrogen, carbon dioxide, xenon, krypton, sulfur hexafluoride, chlorotrifluoromethane, dichlorodifluoro-methane, bromotrifluoromethane, bromochlorodifluoromethane, tetrafluoromethane, dibromo-difluoromethane, dichlorotetrafluoroethane, chloropentafluoroethane, hexafluoroethane, hexafluoropropylene, octafluoropropane, hexafluoro-butadiene, octafluoro-2-butene, octafluorocyclobutane, decafluorobutane, perfluorocyclopentane, dodecafluoropentane, and tetradecafluorohexane.

30. The method of claim 28, wherein the physiologically acceptable gas comprises octafluoropropane.

31. The method of claim 1, wherein the phosphatidylcholine lipid is a 1,2-distearoyl-sn-glycero-3-phosphocholine.

32. The method of claim 1, wherein the average diameter of the microparticles is in the range of 0.5 to 5 μm.

33. The method of claim 1, wherein the microparticle composition is an intermediate microparticle composition.

34. The method of claim 1, wherein a temperature in step d) exceeds the chain melting temperature (Tc) of the lipids in the lyophilisate.

35. The method of claim 1, wherein an initial temperature in step d) is approximately 60° C.

36. A method of preparing a microparticle composition suitable for molecular imaging, the microparticle composition comprising microparticles, wherein the microparticles comprise: a core microparticle structure having a central area and a shell, and wherein the core microparticle structure comprises a phosphatidylcholine lipid, a first phosphatidylethanolamine lipid comprising at least one maleimide moiety, and an alkoxylated fatty acid, the method comprising the steps of: a) forming a lyophilisate comprising the phosphatidylcholine lipid, the first phosphatidylethanolamine lipid, and the alkoxylated fatty acid; b) dissolving the lyophilisate in an aqueous-based solution; c) heating the solution to a temperature at or above a chain melting temperature (Tc) of the lipids in the lyophilisate; and d) shear-mixing the solution in presence of a gas.

37. The method of claim 36, wherein the temperature in step c) is at or above 60 C.

38. The method of claim 36, wherein the temperature in step c) is at or above 65° C.

39. The method of claim 36, wherein the temperature in step c) is in a range of approximately 60 to 65° C.

40. The method of claim 36, further comprising the step of: e) covalently attaching at least one molecular binding element to the shell of the core microparticle structure.

41. The method of claim 36, wherein the core microparticle structure comprises: (i) a C18-24 saturated phosphatidylcholine lipid in a molar ratio of 72 to 78; (ii) a first C18-24 saturated phosphatidylethanolamine lipid comprising at least one maleimide moiety in a molar ratio of 7 to 12 and a polyethylene glycol chain with a molecular weight of at least 500; and (iii) an alkoxylated fatty acid in a molar ratio of 12 to 18, the alkoxylated fatty acid being a C18-24 saturated polyethylene glycol fatty acid ester.

42. The method of claim 36, wherein a temperature in step d) is a temperature at or above the chain melting temperature (Tc) of the lipids in the lyophilisate.

43. The method of claim 36, wherein an initial temperature in step d) is approximately 60° C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0123] The invention will now be described in more detail by way of example only and with reference to the following Figures.

[0124] FIG. 1. Frozen section immunohistochemistry of the mouse heart. Representative example from mice 6 h post lipopolysaccharide (LPS) pre-treatment. Magnification 200×. Low power magnification (low mag) 40×.

[0125] FIG. 2. E-selectin (Esel) targeting microparticles & in vitro validation. (a) A schematic representation of Esel targeting particles. (b) Morphology of Esel targeting particles. (c) Size distribution of Esel targeting particles. Each bar represents mean±SEM, n=5 batches of particles prepared on separate occasions. (d) In vitro validation of Esel targeting particles. Each bar represents mean SEM of attached particle density relative to that of Esel targeting particles on dish E.

[0126] FIG. 3. In vivo validation of the Esel targeting microparticles. Intravital microscopy (IVM) of Esel targeting particles in the mouse cremaster. (a) Elimination of circulating-particles against time in the wild-type (WT) and Esel knock-out (KO) group of mice. Data point represents mean±SD. (b) Accumulation of attached particles against time in 5 WT and 5 Esel KO mice. (c) Silicon intensifier target (SIT) camera images (fluorescence microscopy, magnification 200×) of attached particles in a WT (i) and Esel KO (ii) mice. (d) Quantification of attached particles on the cremaster venular wall. Each point represents one animal; bars represent group mean±SEM. (e) Confocal microscopy of Esel targeting particles in the mouse cremaster venule. Esel in green (i, ii), particles in red (iii, iv), endothelium (PECAM-1) in grey (v, vi). Combined all 3 components (vii, viii).

[0127] FIG. 4. Real-time ultrasound (US) molecular imaging of Esel expression in the mouse heart. (a) Sequential 14 MHz Contrast Pulse Sequencing (CPS) images of the heart in end-diastole parasternal short-axis (PSA) view, in a (1) WT and (2) Esel KO mouse pre-treated with LPS, respectively. TICs of the LV cavity (from region of interest (ROI) C) and myocardium (from ROI M) for the respective animal are shown beneath; each data point represents background subtracted mean signal intensity (I)±SD; suggested bolus (B), distribution (D) and elimination-phase (E) of the time signal intensity curve (TIC) are indicated. (b) PSA, parasternal long-axis (PLA) and apical 4-chamber (A4C) views of the heart at 14 and 7 MHz CPS, >20 min post particle administration (when freely circulating-particles have cleared from the blood pool (left ventricular (LV) cavity)). Animal, gain and MI were the same between both frequencies. Arrow indicates mid anteroseptal wall. Baseline images before particle administration are shown in FIG. 6.

[0128] FIG. 5a. Real-time US molecular imaging of Esel expression in the mouse abdomen. Combined 14 MHz ‘CPS-contrast only’ and ‘B-mode images’ before (i, ii, v, vi) and ≈30 min post (iii, iv, vii, viii) particle administration. Kidney (K), liver (L), spleen (S).

[0129] FIG. 5b. Real-time US molecular imaging of Esel expression in the mouse lower abdomen. Separate 14 MHz ‘CPS-contrast only’ and ‘B-mode images’ before (ix, x, xiii, xiv) and ≈30 min post (xi, xii, xv, vxi) particle administration. Kidney (K), spleen (S).

[0130] FIG. 6. Baseline images (before particle administration) for FIG. 4b.

[0131] FIG. 7. Temporal expression of Esel in the mouse heart. (a) Esel mRNA. Each data point represents one animal. Exponential line of best fit ±95% confidence interval (CI) is shown. (b) Esel cell-surface protein. Each data point represents mean±95% CI for 5, 5, 4, 5 mice at LPS.sub.Time 3, 5, 8, 24 h, respectively. Exponential line of best fit ±95% CI is shown. (c) Esel mRNA vs. Esel cell-surface protein.

[0132] FIG. 8. Acoustic quantification of Esel expression in the mouse heart. Pearsons r: (a) WT=−0.87, KO=−0.08; (b) WT=0.84, KO=not applicable; (c) WT=−0.78, KO=−0.05; (d) WT=0.81, KO=not applicable. Solid circle=WT, open square=Esel KO. Error bar represents SD.

EXAMPLES

[0133] Experimental Details

[0134] Antibodies

[0135] MES-1 monoclonal antibody (mAb), a rat IgG2a,k against mouse Esel,.sup.10 and its F(ab′).sub.2 fragment (MES-1 F(ab′).sub.2) were provided by Dr D Brown (UCB Celltech, UK). AF488-MES-1 (MES-1 labelled with 7 Alexa Fluor® 488 fluorescence dye) and reduced MES-1 F(ab′).sub.2 (2 thiol (SH) groups per F(ab′).sub.2 from tris(2-carboxyethyl)phosphine hydrochloride (TCEP) reduction) were prepared as described below. MEC13.3 mAb, rat IgG2a,k against mouse PECAM-1 (BD Biosciences). Allophycocyanin-labelled mAb against mouse PECAM-1 (BD Pharmingen). Rat IgG2a,k isotype negative control mAb (BD Biosciences). Biotinylated rabbit mAb against rat IgG2a (secondary antibody) (Vector Laboratories). [0136] (a) Reduction of MES-1 F(ab′).sub.2 for microparticle conjugation. MES-1 F(ab′).sub.2 was reduced using 4 molar excess TCEP (Sigma-Aldrich): MES-1 F(ab′).sub.2 (83.3 μM, 8.3 mg/ml) and TCEP (333.3 μM, 0.096 mg/ml) in Exchange Buffer (50 mM 2-(N-Morpholino)ethanesulfonic acid (Sigma-Aldrich), 2 mM ethylenediaminetetraacetic acid (Sigma-Aldrich), pH 6) were incubated for 1 h at 37° C. under constant agitation. The reaction volume ranged 1-1.6 ml. The reaction was stopped by placing on ice and immediate purification of the reduced F(ab′).sub.2 using spin column gel filtration chromatography with a 5 ml-Zeba Desalt Spin Column (size exclusion limit 1,000 Da) according to the manufacturer's instructions (Perbio Science), at 4° C. The spin column was previously equilibrated in cold Exchange Buffer. The degree of reduction of the F(ab′).sub.2 (number of SH per F(ab′).sub.2) was determined spectrophotometrically using Ellman's test with Ellman's Reagent (Perbio Science) according to the manufacturer's instructions with the following modifications: the Ellman's reaction consisted of 2.5 μl Ellman's Reagent (10 mM, 4 mg/ml), 7.5 μl Exchange Buffer and 90 μl of the purified reduced F(ab′).sub.2 in Exchange Buffer, incubated at room temperature (rt) covered with aluminium foil to minimise light exposure; absorbance at 412 nm (A.sub.412) was measured at 24 min from the start of the Ellman's reaction, to determine the concentration of SH in the reduced F(ab′).sub.2 sample by reference to a standard curve of Ellman's reaction with known concentrations of SH-containing compound, L-Cysteine hydrochloride (Perbio Science) in Exchange Buffer (pH6); duplicate Ellman's ‘blank’ reaction where the Exchange Buffer was added in place of the reduced F(ab′).sub.2 was used for baseline subtraction of A.sub.412 from the test samples. The concentration of reduced F(ab′).sub.2 was determined from A.sub.280 in the absence of Ellman's Reagent (with the spectrophotometer zeroed using Exchange Buffer), as the latter would interfere with A.sub.280. The degree of F(ab′).sub.2 reduction was calculated as:

[00001]${\mathrm{SHperF}\left({\mathrm{ab}}^{\prime }\right)}_2=\frac{\left[\mathrm{SH}\mathrm{in}\mu M\right]}{\left[{F\left({\mathrm{ab}}^{\prime }\right)}_2\mathrm{in}\mu M\right]}.$

The reduced F(ab′).sub.2 contained 2 SH groups per molecule of F(ab′).sub.2. The purified reduced F(ab′).sub.2 was kept at concentration of ≈8 mg/ml (80 μM) in Exchange Buffer for subsequent conjugation to microparticles. [0137] (b) Labelling of MES-1 mAb with Alexa Fluor® 488 (AF488) fluorescence dye for confocal microscopy. MES-1 was labelled with Alexa Fluor® 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester, 5-isomer (Molecular Probes) as follows. A 100 μl-labelling reaction mixture consisting of 50 μM MES-1 mAb (25.7 μl at 194 μM or 29.1 mg/ml in phosphate buffered saline (PBS) pH 7.5), 750 μM AF488 (6.6 μl at 11.3 mM or 10 mg/ml in water), 2.6 μl ( 1/10.sup.th volume of MES-1 mAb added) 1M NaHCO3 pH 8.5 and 65.1 μl distilled H.sub.2O, was incubated at rt for 1 h, with gentle manual agitation at 30 min. Controls included no dye or no MES-1 mAb. AF488-labelled MES-1 was then purified from the reaction mixture and suspended in PBS (pH 7.5), using gel-filtration chromatography spin column with 6 kDa size exclusion limit (Bio-Spin P-6 Column with SSC packing buffer, BioRad) according to the manufacturer's instructions with the following specific conditions: the Bio-Spin column was buffer exchanged first with PBS using 3 wash cycles; all centrifugations were carried out at 20° C. The concentration and degree of labelling of AF488-labelled MES-1 was determined using a spectrophotometer. Samples were diluted in PBS to 100 μl volume in duplicates, and absorbance measured at 280 nm (A.sub.280) and 495 nm (A495). The concentration of the labelled mAb was calculated as

[00002]$\left[\mathrm{mAb}\right]\mathrm{in}\mathrm{mg}/\mathrm{ml}=\frac{A_{280}-\left(A_{495}\times 0.11\right)}{1.4}\times \mathrm{dilution}\mathrm{factor},$

where 0.11 is the correction factor for AF488's contribution to A.sub.280. The concentration of the mAb in mg/ml was converted to μM using:

[00003]$\left[\mathrm{mAb}\right]\mathrm{in}\mu M=\frac{\left[\mathrm{mAb}\right]\mathrm{in}\mathrm{mg}/\mathrm{ml}}{150000}\times {10}^6,$

where 150,000 is the molecular weight of mAb in Da, 10.sup.6 is the multiplication factor for converting M to μM. The concentration of AF488 was calculated as:

[00004]$\left[\mathrm{AF}488\right]\mathrm{in}\mu M=\frac{A_{495}}{71000}\times \mathrm{dilution}\mathrm{factor}\times {10}^6,$

where 71,000 is the approximate molar extinction coefficient of AF488 dye (in cm.sup.−1 M.sup.−1) at 494 nm, 10.sup.6 is the multiplication factor for converting M to μM. Finally, the degree of labelling was calculated as:

[00005]$\mathrm{Degree}\mathrm{of}\mathrm{labelling}=\frac{\left[\mathrm{AF}488\right]\mathrm{in}\mu M}{\left[\mathrm{mAb}\right]\mathrm{in}\mu M}.$

Aluminium foil was used at all stages to minimise light exposure. Under this protocol, unreacted AF488 was retained in the column, confirmed by the ‘no MES-1 mAb’ control reaction. The labelling reaction molar ratio of 15:1 (AF488:MES-1) yielded MES-1 labelled with 7 AF488 molecules each, labelling efficiency=46%, yield ≈89%. The purified AF488-labelled-MES-1 (6.748 mg/ml, 44.984 μM) was aliquoted, wrapped in aluminium foil and store at −20° C. until use. The preservation of sensitivity and specificity to Esel of the AF488-labelled MES-1 was confirmed using immunohistochemistry on frozen heart sections of wild-type (WT) and Esel knock-out (KO) mice pre-treated with lipopolysaccharide (LPS).

[0138] Microparticle Preparation

[0139] Native (unconjugated) maleimide-functionalised lipid-shelled octafluoropropane (C.sub.3F.sub.8) microparticles were prepared by sonication of an aqueous suspension containing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC: Avanti Polar Lipids, AL), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide(polyethylene glycol)-2000) (DSPE-PEG.sub.2000-Mal; Avanti Polar Lipids), mono-stearate poly(ethylene)glycol (PEG.sub.40 stearate: Sigma-Aldrich), and fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) at 75:9:14:2 molar ratio, in the presence of C.sub.3F.sub.8. To make Esel targeting particles, TCEP-reduced MES-1 F(ab′).sub.2 containing 2 SH groups per F(ab′).sub.2 were grafted to the shell outer-surface of these native particles by maleimide-thiol conjugation. The conjugation reaction ratio was 4.338×10.sup.6 F(ab′).sub.2 molecules per particle (7.2 nmol F(ab′).sub.2 per 10.sup.9 particles). (NB. The total number of DSPE-PEG.sub.2000-Mal molecules in the aqueous suspension/total number of unwashed particles produced from the aqueous suspension=4.338×10.sup.6. If ≤10% of the components in the aqueous suspension were incorporated onto the particle shell, and the molar ratio of the components on the shell remained close to that of the initial aqueous suspension,.sup.56 then a particle of population mean size would contain ≤4.338×10.sup.5 maleimide molecules, and the estimated F(ab′).sub.2:maleimide conjugation reaction molar ratio would be ≥10:1). The conjugation reaction was carried out for 30 minutes (min) at 4° C., near neutral pH, under CFs atmosphere with constant gentle agitation; the reaction was terminated by adding excess N-Ethylmaleimide (NEM) to quench any unreacted SH. Particles were washed with cold degassed normal saline using multiple cycles of centrifugation flotation under C.sub.3F.sub.8 atmosphere at 4° C. before and after particle conjugation, to remove unincorporated components and particle fragments. Freshly prepared particles were immediately divided into 20-50 μl aliquots, capped and sealed with parafilm (American National Can), then snap frozen in liquid nitrogen and stored at −80° C. until use. The concentration of subsequently thawed Esel targeting particles ranged 1-3×10.sup.9 particles/ml amongst 5 batches prepared at different times.

[0140] Detailed experimental conditions are as follows. Native (unconjugated) microparticles were prepared by dispersing DSPC, DSPE-PEG.sub.2000-Mal, PEG.sub.40 stearate and DiI at molar ratio of 75:9:14:2 in a small amount of cyclohexane:chloroform solvent (1:2), in a 50 ml round-bottomed flask. Excess solvent was extracted using a stream of gaseous nitrogen. The lipid was then transferred to a freeze dryer and lyophilised to full dryness under a reduced atmosphere (e.g., 1.3×10.sup.4 Pa) at −78.5° C. (using a jacket of dry ice). The dry powder (lyophilisate) was then dispersed in a suitable aqueous-based solution (e.g., normal saline or normal saline containing 0.01% propylene glycol (PGNS: propylene glycol 103.5 mg/ml, glycerol 126.2 mg/ml. NaCl 6.8 mg/ml, pH ≈7.4)), to a concentration of 4 mg/ml, homogenised by sonication in an ultrasonic bath at 60-65° C. until transparent. Once fully dissolved, the solution was gently sparged with C.sub.3F.sub.8 gas (F2 Chemicals). Microparticles were then formed using a shear-mixing approach, by sonic dispersion of C.sub.3F.sub.8 using a sonicator (Misonix 3000, QSonica, CT). The probe tip was positioned about 2 mm into the solution and sonication was performed with a high-intensity ultrasound horn (20-21 kHz) for 30-60 s at an acoustic power of approximately 120 W with an initial temperature of approximately 60° C. More C.sub.3F.sub.8 gas was sparged into the microbubble dispersion, and the vessel capped and immediately plunged into ice cold water (3 min) to dissipate the heat generated during the sonication process. Microparticles produced were washed (purified) by centrifugation flotation at 1,000 g 4° C. for 15-25 min, using a Beckman Coulter Allegra X-15R Centrifuge (Beckman Coulter): particles float to the top of the sample vial after centrifugation, the subnatent was removed and replaced with equal volume of cold degassed normal saline (pH 7.4). The wash step was repeated 7 times to remove unincorporated shell components and particle fragments. To produce Esel targeting microparticles, these washed native microparticles were added to reduced MES-1 F(ab′).sub.2 whilst mixing (each reduced F(ab′).sub.2 molecule contained 2 SH groups, prepared as described above). The conjugation reaction ratio used to produce the successful Esel targeting particles was 4.338×10 F(ab′).sub.2 molecules per particle. The concentration of particles and F(ab′).sub.2 in the conjugation reaction mixture ranged 5-8×10.sup.9/ml and 35-60 uM (3.5-6 mg/ml), respectively. The reaction mixture contained approximately 2/3 volume of Exchange Buffer (pH 6) from the reduced F(ab′).sub.2 and 1/3 volume of normal saline (pH 7.4) from the washed particles. The conjugation reaction was incubated at 4° C. for 30 min, continuously mixed gently on a vertically tilted rotating wheel. Particle conjugation was terminated by adding 80 mM NEM (Sigma-Aldrich) dissolved in dry dimethyl sulfoxide (DMSO. Sigma-Aldrich) at 20 molar excess to F(ab′).sub.2—the reaction mixture was incubated at 4° C. for 30-60 min on the rotator. Typically, the concentration of NEM and DMSO in the reaction mixture was ≈1 mM and ≤1.7% v/v, respectively. The particles were then washed 4 times with cold normal saline by centrifugation flotation as described above, at 160 g 4° C. for 5 min. This removed unincorporated F(ab′).sub.2, unreacted NEM, DMSO and particle-fragments. To minimise particle loss, all washes and incubations were performed with the particle concentrations kept high (≥1×10.sup.9 particles/ml), under C.sub.3F.sub.8 atmosphere (to reduce the concentration gradient for diffusion of C.sub.3F.sub.8-gas out of the particles) and at 4° C. (to reduce the rate of gas diffusion). To preserve the particles DiI fluorescent dye, the DiI compound, lyophilisate or particles were protected from light.

[0141] Microparticle Morphology, Concentration, Size Distribution and Charge Analysis

[0142] Particle integrity (spherical morphology, absence of aggregation) was examined under microscopy with the particles placed in a haemocytometer (Reichert Bright-Line Metallized Hemocytometer, Hausserscientific, PA) at e.g., 1:200 dilution in cold normal saline. Particle concentration and size distribution were determined by electrozone sensing in a Coulter Multisizer IIe equipped with a 30 μm-diameter orifice counting tube (Coulter Electronics), according to the manufacturer's instructions. The set-up allowed size detection range 0.72-18 μm, resolution 0.09 μm. For particle charge analysis, the particles were dispersed in 1 ml of 1 mM KCl (pH 7.4) at ≈10.sup.7 particles/ml. Its net charge was determined as the zeta potential by light scattering in a Zetasizer Nano ZS (Malvem Instruments), according to the manufacturer's instructions.

[0143] Targeting Microparticle Binding Assay In Vitro

[0144] 100 μl of Esel targeting or non-targeting (native) particles at 2.5×107 particles/ml were placed on inverted polystyrene petri-dishes coated with 200 μl of recombinant homodimeric mouse Esel protein (R&D Systems) at 7 nM (dish E), or on Esel coated dishes previously blocked with 500 μl of excess MES-1 F(ab′).sub.2 at 67 nM (dish B), or on non-coated dishes where phosphate buffered saline pH 7.5 (PBS) was used instead of Esel for dish coating (dish P). Unattached particles were gently washed off after 1 min. The dishes were then re-filled with cold degassed PBS for immediate examination under an upright light microscope equipped with immersion objective lens. The number of particles attached on each dish was counted and averaged from 10 random optical fields (OFs) to determine the attached particle density.

[0145] The detailed methodology is as follows. Polystyrene petri-dish (Corning® 35 mm Not TC-Treated Culture Dish, Corning Life Sciences) was coated with 200 μl recombinant homodimeric mouse Esel protein (R&D Systems) at 1.25 μg/ml (7 nM, diluted in phosphate buffered saline pH 7.5 (PBS)) for 1 h at rt (dish E). PBS was used instead of Esel as ‘blank’ negative control (dish P). Non-specific binding sites were then blocked with 4 ml bovine serum albumin (BSA, Sigma-Aldrich) at 2.5% w/v in PBS for 2 h at rt. BSA was then discarded and the dish washed with PBS. To prepare Esel coated dish blocked with excess MES-1 F(ab′).sub.2 (dish B), 500 μl MES-1 F(ab′).sub.2 at 6.67 μg/ml (67 nM) was placed in dish E for 30 min at rt, then washed with PBS. Due to the buoyancy of the particles, the dishes were inverted for incubation with particles for 1 min at rt: 100 μl Esel targeting or non-targeting (native) particles at 2.5×10.sup.7 particles/ml (diluted in cold degassed PBS) were used. Unattached particles were then gently washed off and the dishes re-filled with cold degassed PBS for immediate examination using an upright light microscope equipped with immersion objective lens, connected to a camera and monitor. The number of particles attached to each dish was counted and averaged from 10 random optical fields (OFs) on the monitor display, the surface area of the latter determined using a stage micrometer. The attached particle density was thus determined.

[0146] Animals

[0147] Wild-type (WT) mice: adult male C57Bl6/Jax (Charles River, UK). Esel knock-out (KO) mice: adult male Esel homozygote KO on C57Bl6 background,.sup.57 bred locally from mice donated by Dr K Norman and Prof P Hellewell (University of Sheffield, UK). All the animal work was carried out under Project Licences and Personal Licences granted by the Home Office under the Animals (Scientific Procedures) Act 1986; ethical approval was additionally obtained from the local Ethical Review Panel.

[0148] Lipopolysaccharide (LPS) Mouse Model (Experimental Endotoxaemia)

[0149] WT and Esel KO mice were pre-treated with 50 μg LPS from E. coli 0111:B4 (Sigma-Aldrich), made up to 200 μl volume in normal saline, by intraperitoneal (ip) injection to induce systemic inflammation. Systemic administration of LPS by ip injection produces systemic inflammation, which includes induction of Esel expression in multiple organs including the heart and kidneys..sup.58, 59 This animal model differs from others used in particle-targeting (such as ischaemia-reperfusion injury, heart transplant rejection, thrombosis, chronic-ischaemia/tumour angiogenesis animal models) in that: (i) it does not require surgical procedures which may introduce confounding variables such as surgical trauma or blood clots; (ii) the particles target molecules present in multiple organs (not just one), uniquely allowing assessment of targeting particle specificity in multiple organs simultaneously, and assessing the imaging technique's ability to detect target molecule in a tissue of interest in the presence of ‘particle steal’ by other tissues: and (iii) the target molecule expression in the myocardium was essentially global and uniform, uniquely allowing the study of targeted particle signal attenuation. Additionally, the combination of Esel being solely expressed on activated endothelial cells and its essentially global uniform expression in the myocardium of LPS pre-treated mice, makes the LPS mouse model-Esel combination an ideal in vivo model for testing acoustic quantification of molecular expression.

[0150] Immunohistochemistry

[0151] Immunohistochemistry was performed on acetone-fixed cryosections of freshly harvested hearts of WT (with/without LPS pre-treatment) and Esel KO (pre-treated with LPS) mice. After blocking non-specific binding sites with 100 μl of 1:1000 rabbit serum (Sigma-Aldrich) for 1 hour (h) at room temperature (rt), sections were incubated for 1 h at rt with 100 μl of 0.01 mg/ml primary antibody: MES-1 (for Esel), MEC13.3 (for PECAM-1, endothelial marker) or isotype negative control. Each section was then incubated with 100 μl of 0.005 mg/ml biotinylated secondary antibody for 60 min at rt. After blocking of endogenous peroxidase with 0.3% H.sub.2O.sub.2 methanol for 20-30 min at rt, the horseradish peroxidase-based detection system. Vectastain ABC kit (Vector Laboratories), was used with 3,3′-Diaminobenzidine solution (SIGMAFAST™ DAB tablet, Sigma-Aldrich) as the chromagen substrate. Sections were counterstained using Harris Modified Hematoxylin Solution (Sigma-Aldrich) and 1% NaHCO.sub.3, then dehydrated through 70-100% ethanol, dried and mounted with Histomount (VWR), and examined under light microscopy. The duration between the time of LPS pre-treatment and sacrifice of the animal for immediate tissue harvesting was noted as the LPS.sub.Time.

[0152] Reverse Transcriptase-Real Time Quantitative Polymerase Chain Reaction (RT-qPCR). WT mice were pre-treated with LPS as described above. The duration between the time of LPS pre-treatment and sacrifice of the animal for immediate tissue harvesting was noted as the LPS.sub.Time. Freshly harvested tissues were kept in RNAlater®, solution (Ambion) to preserve ribonucleic acid (RNA) in-situ; total RNA was subsequently extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand complementary deoxyribonucleic acid (cDNA) synthesis was then performed using the Qiagen Omniscript® Reverse Transcription kit (Qiagen) according to the manufacturer's instructions. This was followed by real-time qPCR with the SYBRGreen detection method for Esel and hypoxanthine phosphoribosyltransferase-I (HPRT-I), carried out on a 96-well plate in the iCycler™ (iCycler iQ Real-Time PCR Detection System, Bio-Rad) according to the manufacturer's instructions. All PCR reactions were carried out in triplicate wells on the same plate. The primer sequences were: Esel forward primer 5′-CTCATTGCTCTACTTGTTGATG-3-, Esel reverse primer 5′-GCATTTGTGTTCCTGATTG-3′, HPRT-I forward primer 5′-ATTAGCGATGATGAACCAG-3′, HPRT-I reverse primer 5′-AGTCTTTCAGTCCTGTCCAT-3′. For data analysis, the threshold cycle (C) was determined from the amplification plot using the iCycler™ iQ Optical System Software Version 3.0a (Bio-Rad). As PCR efficiency of the Esel and HPRT-I primer pairs differed by <5% (93±4% and 92±3% (mean±SD), respectively; n=4 each), comparative Ct method was used to estimate the amount of Esel messenger (mRNA) relative to that of HPRT-I, using the formula: Esel mRNA(% HPRT-I)=2.sup.−ΔCt, where ΔCt=C.sub.Esel−C.sub.HPRT-I, subscripts refer to the gene of interest. Mean of the replicates was used and plotted against LPS.sub.Time for each animal. Further details of the methodology are as follows. The yield of total RNA from the mouse heart was typically ≈1 μg pure RNA per 1 mg tissue, kept at concentrations over ≈1 mg/ml in molecular grade (RNase-free) H.sub.2O (Sigma-Aldrich). The RT reaction mixture for first-strand cDNA synthesis consisted of 1 μg total RNA, 2 μl 10× buffer RT, 2 μl deoxyribonucleotide triphosphate (dNTP) mix (5 mM each 2′-deoxyadenosine 5′-triphosphate (dATP), 2′-deoxycytidine 5′-triphosphate (dCTP), 2′-deoxyguanosine 5′-triphosphate (dGTP), 2′-deoxythymidine 5′-triphosphate (dTTP), 1μ (4 units) Omniscript reverse transcriptase, 2 μl (1 μg) oligo(dT).sub.12-18 primer (Invitrogen) and molecular grade H.sub.2O made up to a total reaction volume of 20 μl, incubated for 1 h at 37° C. qPCR was carried out in a 25 μl-reaction volume in each well of a 96-well 0.2 ml thin-wall PCR plate (Bio-Rad) covered with an Optical Quality Sealing Tape (Bio-Rad). The qPCR reaction mixture consisted of 5 μl cDNA template (1:50 water dilution of the finished RT reaction), 0.5 μl (10 μM) each of the forward and reverse primer for the respective gene (see text for primer sequence: the primers were custom ordered from Invitrogen), 6.5 μl molecular grade H.sub.2O and 12.5 μl iQ™ SYBR® Green Supermix (Bio-Rad). The qPCR cycling condition was: initial 3 min denaturing step at 95° C. (Well Factor analysis in first 90 s): then 40 cycles of 15 s at 95° C., Imin at 56° C.; melt-curve analysis in 0.5° C. steps (Imin denaturation at 95° C., Imin reset at 56° C., then 80 cycles of 10 s at 60° C. with 0.5° C. increment for each cycle); final cooling step at 4° C. Esel and HPRT-1 were amplified on the same plate for each animal: no-template negative control using molecular grade H.sub.2O in place of cDNA template for both primer pairs were included in all plates. For data analysis, wells with abnormal amplification plot or melt-curve were excluded.

[0153] Intravital Microscopy (IVM) Set-Up

[0154] Inflammation of the cremaster muscle was produced by intrascrotal injection of 50 ng recombinant IL-1β (R&D Systems) 2 h before surgery in WT and Esel KO mice. The tail vein was cannulated with a 24G 0.7×19 mm intravenous (iv) catheter (dead space ≈50 μl) (BD Medical). Long duration anaesthesia was achieved using intraperitoneal (ip) injection of 200-300 μl mixture containing 1 mg/ml xylazine (Rompum, Bayer) and 10 mg/ml ketamine hydrochloride (Ketalar, Parke-Davis) in normal saline. The animal was placed on a custom-built thermo-controlled (37° C.) IVM stage. Under a dissection microscope, the right or left testis was gently exteriorised through a scrotal incision. A longitudinal incision was made along the cremaster muscle, which was then spread out and pinned down across a translucent microscopy stage. The exteriorised muscle was maintained by continuous super-fusion of thermo-controlled Tyrode's Salt buffer solution (9.6 g Tyrode's Salts (Sigma-Aldrich)+1 g sodium hydrogen carbonate, made up to 1 L volume with sterile distilled water). Observations and recordings were made using an upright microscope (Axioskop, Carl Zeiss) equipped for bright field and fluorescence microscopy, with 20× and 40× immersion objective lens (Water Achroplan, Carl Zeiss), a charge coupled device (CCD) camera (Color Chilled 3CCD Camera with controller, Hamamatsu Phototonics), a silicon intensifier target (SIT) camera (C-2400-08, Hamamatsu Photonics), a monitor (Triton, Sony), a S-VHS recorder (Model AG 6730 SVHS 625, Panasonic) and a Personal Computer. To compare shear rates against particle attachment between WT and Esel KO animals, an Optical Doppler Velocimeter (Microcirculation Research Institute, Texas A&M University, Texas), was used to measure microvascular centreline red blood cell velocities (V.sub.rbc) in 20-40 μm diameter venules of the WT and KO mice, before particle injection. 5 random vessel-segments were measured per animal.

[0155] IVM of Esel Targeting Microparticles in the Mouse Cremaster

[0156] 1.5×10.sup.7 Esel targeting particles in 100 μl normal saline were administered as a rapid iv bolus through a tail vein catheter, immediately followed by a 100 μl normal saline flush, for intravital microscopy assessment in the cremaster of anaesthetised (xylazine/ketamine mixture ip) WT and Esel KO mice. All animals were pre-treated with 50 ng recombinant IL-1β (R&D Systems) intrascrotally, 2 hours (h) before exteriorization of the muscle, to induce cremaster inflammation. Observations were made using an upright microscope equipped for bright field and fluorescence microscopy, with 20× and 40× immersion objective lens, CCD and SIT cameras. To determine the shear rates against particle attachment, V.sub.rbc were measured in 20-40 μm diameter venules from 5 random vessel-segments per animal, using an Optical Doppler Velocimeter before particle injection. All recordings were made using a S-VHS recorder and Personal Computer. All animals were sacrificed at the end of the experiment. See above for details of the set-up. Blood flow and particles were assessed over several OFs encompassing a number of different vessels under bright field and fluorescence microscopy. The number of freely circulating-particles in an OF on the monitor were counted over 10 s under fluorescence microscopy at 5, 7, 10±15 min after particle injection. The accumulation of attached particles (defined as not moving for >3 s) in an OF field were assessed for up to 15 min post particle injection. The number of particles attached to 20-40 μm diameter venules were counted at ≈15 min after particle injection (when freely circulating-particles were absent/minimal) from 1-5 400 μm length segments per venule, 2-6 venules per animal. In some animals, the attached particles in the same OF were followed-up for up to 90 min using intermittent combined bright field and fluorescence microscopy, looking for the presence/absence of transmigration into the tissue interstitium or cellular internalisation. For analysis, the density of attached particles was expressed as the number of particles per vessel surface area (VSA), where VSA(mm.sup.2)=πD(mm)×L(mm), D and L were the vessel segment diameter and length, respectively. The attached particle density for each venule was taken as the mean of its segments, and that for each animal was taken as the mean of its venules. Shear rate was estimated from V.sub.rbc as:

[00006]$\mathrm{Shear}\mathrm{rate}\left(s^{-1}\right)=8\frac{V_b\left(\mathrm{cm}/s\right)}{D\left(\mathrm{cm}\right)},\mathrm{where}{V}_b\left(\mathrm{cm}/s\right)=\frac{V_{\mathrm{rbc}}\left(\mathrm{cm}/s\right)}{\alpha },$

V.sub.b is the mean bulk velocity, α the factor converting V.sub.rhc to V.sub.b, taken as 1.6 for Poiseuille flow in veins..sup.60 The shear rate for each animal was taken as the mean of the 5 random venule segments sampled.

[0157] Confocal Microscopy of Esel Targeting Microparticles in the Mouse Cremaster

[0158] 1.5×10.sup.7 Esel targeting particles were administered to WT and Esel KO mice pre-treated 2.5 h beforehand with 50 ng IL-1β intrascrotally, using methods described for IVM above. 15 min later, a rapid iv bolus of 150 μl cocktail containing 50 μg AF488-MES-1 (for Esel) and 25 μg allophycocyanin-labelled mAb against mouse PECAM-1 (endothelial marker) in normal saline was administered iv, followed by a 100 μl normal saline flush. After a further 15-20 min, animals were given terminal anaesthesia using iv bolus of a xylazine/ketamine mixture, followed by perfusion with PBS to remove unattached particles and mAb in the circulation. Immediately thereafter, the cremasters were harvested and fixed in 4% paraformaldehyde PBS for confocal microscopy (z-stacked). 3 different fluorescence, DiI (particles), Alexa Fluor) 488 (Esel) and allophycocyanin (PECAM-1) were scanned in series at each depth before moving on to the next depth in the z-axis, using an upright confocal laser-scanning microscope (LSM 5 PASCAL, Carl Zeiss) with a 40× immersion objective lens. Images were processed using Zeiss LSM 5 Image Browser software.

[0159] Further details of the methodology are as follows. Following the iv administration of Esel targeting particles and antibody cocktail containing AF488-MES-1+allophycocyanin-labelled mAb against mouse PECAM-1, unattached particles and mAb in the circulation were removed by perfusion with PBS. This was achieved by exposing the heart and upper abdomen by dissection. A snip incision was then made in the right atrium followed by injection of PBS into the LV cavity using a needle connected to a 20 ml-syringe. This allowed the PBS to perfuse the body, it and the blood left the circulation via the incision in the right atrium. Adequate PBS perfusion was assumed when the liver turned pale due to the replacement of blood by PBS. The cremaster muscle was then harvested immediately, spread out and fixed in 4% paraformaldehyde PBS solution for 30 min at rt, then placed in cold PBS at 4° C. for 5 min. Aluminium foil was used to minimise light exposure to the tissue. Fresh tissues were examined immediately under confocal microscopy (z-stacked), using an upright confocal laser-scanning microscope (LSM 5 PASCAL, Carl Zeiss) with a 40× immersion objective lens (Water Achroplan, Carl Zeiss). 3 different fluorescence, DiI for particles (excite at 543 nm, detect at 560-615 nm), Alexa Fluor® 488 for Esel (excite at 488 nm, detect at 505-530 nm) and allophycocyanin for PECAM-1 (excite at 633 nm, detect at 650 nm) were scanned in series at each depth before moving on to the next depth in the Z-axis. Images were processed using accompanying software, Zeiss LSM 5 Image Browser.

[0160] Ultrasound (US) Imaging

[0161] WT and Esel KO mice were all pre-treated with LPS, tail vein cannulated and anaesthetised with xylazine/ketamine mixture as described above. The chest, abdomen and pelvis were then shaved and the animal placed supine. ECG electrode pads (Ambux: Blue Sensor P, Ambu) were applied to the paws and connected to the US machine (Acuson Sequoia® 512 US system, Siemens, CA) equipped with ‘Small Animal ECG Filter’. A layer of warm gel (Gel for ultrasonic & electrical transmission, Henleys Medical) was coupled between the skin and US transducer (15L8-s linear array transducer, foot print 26 mm, Siemens). US settings used were: 14 MHz (P14 MHz, spatial resolution ≈0.2 mm) Contrast Pulse Sequencing (CPS) mode, transmission power 9 dB giving low mechanical index (MI) 0.22-0.26 estimated by the scanner, dynamic range 55 dB, time gain 0%, CPS gain 8, fundamental 2D gain 15 dB, colour map M:3 (particle signal presented in heated object scale (‘CPS-contrast only’ images), tissue signal in grey scale (‘B-mode’ images)), TEQ was not used. Before particle injection, baseline parasternal short axis (PSA) view at the papillary muscle level, parasternal long axis (PLA) and apical 4-chamber (A4C) views of the heart with and without ‘regional expansion selection’ (RES; giving magnified images with enhanced resolution) were recorded as 3 s-digital clips. Thereafter, imaging was maintained in the PSA view with the transducer fixed in position using a free standing clamp. A stopwatch was then started and 1×10.sup.8 Esel targeting particles (in 100 μl volume made up with normal saline) injected at 10 s via the tail vein catheter as a rapid iv bolus over 1-2 s, followed by a 100 μl-normal saline flush over 1-2 s at 20 s. Continuous US insonation was applied without pausing from time 0-Imin 23 s on the stop-watch, then paused, then resumed only for 3 s each time for digital image acquisition. 3 s-digital clips (RES activated) of the heart containing several consecutive cardiac cycles were recorded at 10 s and 13 s, then at 10 s intervals from 20 s-1 min 20 s, then at 1 min intervals from 2 min 20 s-10 min 20 s, then at 2 min intervals from 12 min 20 s-30 min 20 s, then at 5 min intervals until 60 min 20 s on the stopwatch (image acquisition was stopped earlier if particle contrast enhancement in the left ventricular (LV) cavity (central blood pool) was no longer visible). Unmagnified (non-RES) images of the thorax containing the heart in the PSA view and surrounding tissues were recorded at ≈5 min intervals. Other views of the heart (PLA and A4C views) were acquired at the end. In some animals, 7 MHz (P7 MHz, spatial resolution ≈0.4 mm) CPS imaging at MI 0.22 (gain and other settings kept the same as 14 MHz imaging) was also acquired at baseline and the end of the 14 MHz imaging study. When switching from 14 MHz to 7 MHz CPS imaging, the transmit power was first reduced from −9 dB to −19 dB before reducing the US frequency, to avoid an increase in MI (up to ≈0.7) causing inadvertent particle destruction. In some animals, imaging of other tissues in the thorax, abdomen and pelvis were performed in the antero-posterior projection at baseline and at the end of the cardiac imaging study, to look for particle retention in extra-cardiac tissues. To do this, the probe was positioned transversely and moved slowly caudally from just below the neck to the pelvis during 3 s-image acquisitions: non-RES images in 14 and 7 MHz CPS were acquired. All animals received only one dose of particles to avoid carry-over effect from previous particle dosing (e.g., blocking of Esel binding sites). At the end of imaging, animals were sacrificed and tissues immediately harvested for frozen section immunohistochemistry and qRT-PCR as described above.

[0162] Time-Signal Intensity Curve (TIC) Generation

[0163] Videodensitometric method was used to quantify particle signal intensity off-line, using the YABCO® software (LLC Charlottesville. Va.). End-diastolic image frames of the heart in the PSA view (‘CPS-contrast only’ images) were selected and aligned, those that could not be aligned (e.g., due to large movement artefact) were excluded. Regions of interest (ROIs) were placed on the mid-anterior wall of the myocardium (M) and adjacent region in the LV cavity (C), as shown in FIG. 4a. These regions were chosen because they were consistently least or minimally affected by US attenuation in all animals. The video signal intensities (VI) were ‘linearised’ by log-decompression using the formula: Linearised VI=255×10 (VI−255/255×dB/20), where dB is the dynamic range (55 dB in this study). Linearised VI (I) was expressed in arbitrary acoustic units (AU). I of several end-diastolic image frames within the 3 s-recording period at each time point were averaged, then corrected for background noise by subtracting away average I of the baseline images (images before particle administration) in the respective animals. TICs of the myocardium (tissue) and LV cavity (central blood pool) were constructed by plotting background-subtracted I of the myocardium and LV cavity, respectively, against time post particle administration.

[0164] Acoustic Quantification of Esel Expression and Particle Half-Life In Vivo

[0165] Baseline-subtracted bubble signal intensity in the myocardium at 20 min 10 s post microparticle administration (R20), obtained as per TIC above, was used for analysis. An alternative novel method based on quantitative analysis of the TIC (TIC-based method) was also used to quantify the level of Esel expression. The TIC-based method also allowed other variables to be determined simultaneously, including the retained- and circulating-microparticle half-life in vivo. The LPS.sub.Time for US molecular imaging was taken as the duration between the time of LPS pre-treatment and administration of the targeting microparticles. The mean heart rate (HR) for each animal was calculated from all HRs recorded at different time points during cardiac imaging.

[0166] Statistics

[0167] Pearson correlation, linear or non-linear regression analysis was performed as indicated. Student's t-test or ANOVA with Turkey's post-hoc analysis was used for significance testing where indicated, with p<0.05 taken as significant.

[0168] Results

[0169] Esel Expression

[0170] Frozen section immunohistochemistry showed that Esel was expressed in the heart of all WT mice pre-treated with LPS (n=35, LPS.sub.Time=4-9 h). The spatial distribution was essentially uniform throughout the myocardium but limited to the post-capillary venules and capillaries. Esel was not detectable in the negative controls: WT mice not treated with LPS (n=5), and Esel KO mice pre-treated with LPS (n=10, LPS.sub.Time=4-7 h), FIG. 1.

[0171] Quantification of Esel expression by qRT-PCR. RT-qPCR showed that the concentration of Esel mRNA in the heart decreased exponentially with time after ≈3 h post LPS pre-treatment, reaching very low levels by ≈9 h (n=42, LPS.sub.Time=3.2-15.7 h), FIG. 7a. This trend was similar to that of the cell-surface Esel protein concentration (expressed as % injected dose of radioactivity/g tissue (% ID/g tissue)) determined using iv radio-labelled mAb by Eppihimer el al..sup.58 using the same strain, sex, and age of mice, as well as the same dose and route of LPS administration (n=19), FIG. 7b.

[0172] From the best-fit curves of Esel mRNA concentration vs. LPS.sub.Time (mRNA(in % HPRT-I)=3600e.sup.−0.13 LPS.sup.Time.sup.(in hours), R.sup.2=0.75) and that of Esel cell-surface protein concentration vs. LPS.sub.Time (protein(in % ID/g tissue)=1.21e.sup.−0.13 LPS.sup.Time.sup.(in hours)+0.07, R.sup.2=0.88), using LPS.sub.Time as the common denominator, an empirical formula describing the relationship between the concentration of Esel mRNA and cell-surface protein could be derived as:

[00007]$\mathrm{Protein}\left(\mathrm{in}\%\mathrm{ID}/g\mathrm{tissue}\right)=1.21{\left(\frac{\mathrm{mRNA}\left(\mathrm{in}\%\mathrm{HPRT}-I\right)}{3600}\right)}^{\frac{0.13}{0.7}}+0.07,$

FIG. 7c. Thus the Esel cell-surface protein concentration was predictable from its mRNA concentration in the heart, in this LPS mouse model. Within the mRNA or protein concentration range (55-238% HPRT-1 or 0.63-0.80% ID/g tissue, respectively) used for US quantification of Esel expression in this study (see later), the relationship between the concentration of mRNA and cell-surface protein was approximately linear, allowing direct use of the mRNA concentration as a surrogate quantifier for the cell-surface protein concentration (the latter being the actual target of the targeting microparticles).

[0173] Esel Targeting Microparticles

[0174] A schema of the Esel targeting particles engineered is shown in FIG. 2a. These particles showed spherical morphology: particle-particle aggregation or cross-linkage was minimal, FIG. 2b. The particle size distribution was reproducible amongst 5 batches prepared on separate occasions; particle diameter=2.2(mean)+0.2(SEM) μm, 98.6% or 100% of the particles were under 6 or 10 μm in diameter, respectively. FIG. 2c. The particles (washed) had a near neutral charge, zeta potential approximately 5 mV at pH 7.4.

[0175] The particles were sufficiently echogenic, stable, lacked non-specific binding, and produced no immediate adverse effects in vivo. Particles made using other compositions did not exhibit all of these desired properties. The suitable native particles also contained enough maleimide groups for conjugating sufficient targeting elements onto the particle surface for efficient target binding under flow conditions. The conjugation reaction ratio used to produce the successful Esel targeting particles was 4.338×10.sup.6 F(ab′).sub.2 molecules per particle. Lower F(ab′).sub.2:particle reaction ratio 1×10.sup.6:1 produced particles that could only attach to target under static conditions. It was also found that by keeping the conjugation sites on each binding element to a minimum (e.g., reducing only 1 interchain disulfide bond to produce 2 SH per F(ab′).sub.2), and inactivating any unreacted ones after particle conjugation (e.g., alkylation of unreacted SH), significant particle aggregation due to cross-linking could be avoided. Particles targeting other molecules can be readily made in the same way by substitution with suitable targeting ligands. The use of F(ab′).sub.2 instead of whole mAb in this case eliminated any Fc-medicated non-specific interaction or immunogenic side effects.

[0176] The site-directed maleimide-thiol conjugation of targeting ligands to particles described herein is a departure from the immunogenic (strept)avidin-biotin conjugation chemistry traditionally used in preparing targeting particles. Although other conjugation chemistries can be used, the site-directed maleimide-thiol conjugation method was advantageous due to its low immunogenicity, strong and rapid thioether bond formation at near neutral pH (bond strength in the order of nanonewtons; second-order rate constant 0.8-1×10.sup.4 M.sup.−1 s.sup.−1). The near neutral pH was advantageous in avoiding negative impact on the binding elements and particles during preparation, and prevents dissociation of the binding elements from the particle shell in vivo. Targeting particles based on similar conjugation chemistry, including non-covalent conjugation of targeting ligands to a phospholipid species before particle assembly, exist but only a few are progressing to ultrasound molecular imaging in vivo..sup.44-47, 61-64 none of which showed all of the following desirable attributes which differentiate our microparticles from them: (i) high target binding specificity with proven minimal non-specific retention in remote non-RES tissues not expressing the target molecule, following iv administration; (ii) effective for real-time US molecular imaging; and (iii) effective for acoustic quantification of molecular targets to a high quantitative degree.

[0177] In Vitro Validation of Esel Targeting Microparticles

[0178] Esel targeting particles attached to Esel on coated dish (dish E): 2060(mean)±1070(SEM) particles/mm.sup.2, n=5, FIG. 2d. Specificity of the targeting particles against Esel was demonstrated by their minimal attachment to Esel previously blocked with excess anti-Esel antibody (dish B): 8(mean)±4(SEM) % of targeting particles on dish E, n=5. Additional negative controls using targeting or non-targeting (native) particles on dish not coated with Esel (dish P), or non-targeting particles on dish E and B showed similar low levels of particle attachment: targeting particles on dish P (9±3%, n=5), non-targeting particles on dish E (10±9%, n=2), B (7±7%, n=2) and P (7±7%, n=2). ANOVA with Turkey's post-hoc analysis showed significant differences in the relative density of attached particles between targeting particles on dish E and the negative controls (p<0.0001); no significant difference was observed amongst the negative controls.

[0179] In Vivo Validation of Esel Targeting Microparticles

[0180] Esel targeting particles were administered to 5 WT (body weight: 25(mean)±2(SD) g, range 23-27 g)) and 5 Esel KO (24±2 g, range 22-27 g) mice at 3:06-4:03 h and 3:17-4:30 h post IL-1β pre-treatment, respectively. The particles were seen to circulate and reach the cremaster muscle ≈7-17 s post iv bolus administration through the tail vein. The number of freely circulating-particles decreased with time, their clearance from the blood pool occurred sooner in the WT than KOs, FIG. 3a. Beyond 10 min after particle administration, the number of freely circulating-particles in the blood pool was minimal or undetectable in all animals. The particles attached and accumulated on the cremaster venular wall of the WT animals (370(mean)±46(SEM) particles/mm.sup.2 VSA, n=5 animals); this was minimal in the KOs (11±3, n=5), p <0.0001 (student's t-test), FIG. 3c-d. Particle accumulation on the cremaster vessel wall plateaued soon after particle-bolus administration, FIG. 3b. The accumulated particles persisted beyond the clearance of freely circulating ones from the blood pool. Rolling was observed in a small minority of particles; complete detachment of the attached particles was infrequently seen. Intravascular obstruction by the particles was not observed. Transmigration into the tissue interstitium or cellular internalisation of the attached particles were not detected, when observed intermittently for up to 90 min. Confocal microscopy showed that the attached particles co-localised with Esel expressed on the endothelial cell surface of the WT cremaster venule (n=3 animals, body weight 21.8-24 g); Esel expression was not detectable in the KOs (n=2, 25.7-28.4 g), FIG. 3e.

[0181] Shear rates in the 20-40 μm diameter venules, determined from 3 WT (body weight: 26(mean)±2(SD) g, range 25-28 g) and 3 KO (27±1 g, range 26-29 g) mice, were higher in the WT (329(mean)±46(SEM) s.sup.−1, n=3) than KO group (211±37 s.sup.−1, n=3), probably due to smaller vessel diameters sampled in the former (WT 30(mean)±3(SEM) μm, n=3: KO 34±1 μm, n=3). However, these differences were not statistically significant, p=0.30 and 0.12 (student's t-test) for shear rate and vessel diameter group comparison, respectively.

[0182] Ultrasound Molecular Imaging [0183] (1) Animals. 15 WT (age 5.7(mean)±0.2(SD) weeks, range 5.1-6.1 weeks: body weight 19.5(mean)±1.5(SD) g, range 17-22 g) and 8 Esel KO (age 7.9±3.2 weeks, range 5-13.6 weeks; body weight 22.3±3.7 g, range 17-28 g) mice were imaged. The duration between LPS injection and administration of targeting particles (LPS.sub.Time) ranged 3:26-5:59 h for the WT and 4:27-5:39 h for the KO group. [0184] (2) Cardiac imaging. Non-linear CPS artefacts (orange colour) present both before and after particle administration could be seen in WT and Esel KO animals; these artefacts were small and outside the myocardium, FIG. 4a (frame 0:10). FIG. 4a2 (frame 20:20). Following iv particle-bolus administration, particle signal was first detected in the right heart chambers within 4 heart beats (≈1 s), the signal intensity rose rapidly. This was followed by the appearance of particle signal in the left heart chambers as the particles returned from the pulmonary circulation. Particle signal intensity in the LV cavity and myocardium peaked within 6-7 heart beats (≈1.5-2 s) and 9-12 heart beats (≈2-3 s) after particle administration, respectively. Particle signal intensity then decreased in the heart chambers and myocardium over time, FIG. 4a. Significant US attenuation due to high particle concentration occurred early following particle-bolus administration, with major loss of signal in regions of the heart located distally in the US path (e.g., 5-10 o'clock positions in the myocardium and adjacent LV cavity in the PSA view, FIG. 4a). As the circulating-particle concentration in the blood pool (LV cavity) decreased over time, the attenuation diminished (compare frame 0:30 vs. 6:20, FIG. 4a) but did not disappear (frame 20:20, FIG. 4al)—most likely due to attenuation caused by overlying bone, lung air±retained-particles. Importantly, Esel expression in the myocardium was visualised in real-time, indicated by the persistence of particle signal in the WT myocardium as the freely circulating-particles in the blood pool (LV cavity) decreased and disappeared over time. In the KO myocardium, the persistence of particle signal was low/minimal, consistent with a low/minimal degree of non-specific particle retention in the myocardium, FIG. 4a-b. US attenuation caused by overlying bone, lung air±retained-particles located proximally in the US path, affected certain parts of the myocardium depending on the imaging scan plane—this caused pseudo-loss of targeted particle signal for Esel in the WT animals (e.g., there was artefactual loss of retained-particle signal in the mid-posterior, -inferior, -posteroseptal and -anteroseptal walls of the LV (anti-clockwise from 5-10 o'clock positions, respectively) in the PSA view with 14 MHz CPS imaging). However, by changing the scan plane (e.g., from PSA to PLA or A4C view) to alter the relative position of overlying entities, or by lowering the US frequency to increase its penetrative depth (e.g., from 14 to 7 MHz), these attenuation effects could be overcome with good recovery of the retained-particle signals, FIG. 4b. The global expression of Esel in the WT myocardium was thus demonstrated on US imaging, consistent with the immunohistochemistry data. [0185] In the above situation, US detection of molecular target was limited by late distal attenuation from overlying bone/air and retained-particles located proximally in the US path. However, it was found that such attenuation could be overcome by using lower frequency US (greater penetrative depth) or a different imaging angle. From the human imaging perspective, where the use of lower frequency US (e.g., 3-7 MHz) and multi-plane imaging are the norm, and the footprint of the transducer is much smaller relative to the body size (making it easier to achieve optimal probe position/angle and avoid overlying bone/air), these attenuation issues are likely less important. Nevertheless, refinements in the machine's attenuation correction algorithm may further minimise the attenuation artefacts. [0186] (3) Imaging of other tissues. Comparison of the thoracic, abdominal and pelvic scans between the WT and Esel KO animals with reference to the baseline images (before particle administration), showed that non-specific retention of the targeting particles in non-RES tissues was low/minimal. Esel expression was detected in the renal cortex of WT but not KO animals (FIGS. 5a & 5b). Immunohistochemistry and confocal microscopy confirmed that Esel was expressed predominantly on endothelial cells of renal glomeruli, the latter concentrated in the renal. As expected, the targeting particles were taken up by the spleen and liver in both WT and KO animals (FIGS. 5a & 5b), the major RES tissues involved in particle elimination. [0187] (4) Adverse effects. No death or significant adverse events attributable to the Esel targeting particles were observed. No significant particle-medicated intravascular obstruction in the myocardium, causing loss of regional myocardial perfusion manifest as regional wall motion abnormality, was detected.

[0188] Ultrasound TIC of the Myocardium (Tissue) and LV Cavity (Central Blood Pool)

[0189] Three phases with distinct characteristics were discernible from the TICs following iv bolus administration of the targeting particles, FIG. 4a: [0190] (1) Bolus-phase (B): lasting a few seconds, characterised by an initial rapid rise in particle signal intensity reaching peak or saturating/attenuation levels (as particle concentration increased) within seconds of bolus injection. [0191] (2) Distribution-phase (D): lasting up to 1-2 min (takes a few circulatory cycles to complete), characterised by a short rapid decrease in particle signal intensity, as particles dilute by mixing and distribution to tissues. High particle concentration resulted in signal saturation/attenuation, obscuring re-circulation peaks. As the particle concentration decreased further over time, the particle signal intensity was frequently observed to paradoxically increase as attenuation decreased, giving rise to a second lower peak within 0.5-1 min of particle administration (arrow in insets of FIG. 4a). [0192] (3) Elimination-phase (E): lasting several minutes, characterised by a long slow decrease in particle signal intensity. Particle signal intensity in the LV cavity (representing freely circulating-particle concentration in the central blood pool) became undetectable sooner in the WT than Esel KO animals; in both groups they were essentially undetectable by 20 min post particle administration. In the myocardium, particle signal intensity of WT animals decreased slower than that of KOs, and persisted beyond the disappearance of particle signal in the LV cavity. In KO animals, particle signal in the myocardium became undetectable before the disappearance of particle signal in the LV cavity (as expected for relative myocardial blood volume (rMBV) ≤24%), except when a detectable degree of non-specific particle retention was present.

[0193] Acoustic Quantification of Esel Expression [0194] (1) Animals. 12 WT (age 5.7(mean)±0.3(SD) weeks, range 5.1-6.1 weeks: body weight 19.7(mean)±1.4(SD) g, range 18-22 g) and 8 Esel KO (age 7.9±3.2 weeks, range 5-13.6 weeks; body weight 22.3±3.7 g, range 17-28 g) mice were used. LPS.sub.Time ranged 3:53-5:59 h for the WT and 4:27-5:39 h for the KO group. Excluded from quantitative analysis here were 3 WT animals because of: (i) microparticle dosing error (1 animal); (ii) uncertainties regarding Esel expression level (2 animals); and (iii) 1 of these 3 animals has a TIC that could not be adequately quantified, possibly due to severe attenuation artefact. [0195] (2) Single late time point based method. In both WT and Esel KO groups, freely circulating-particle signal in the blood pool (LV cavity) was absent/minimal by ≈20 min following iv bolus administration of 10.sup.8 particles. Therefore, background-subtracted I in the myocardium at 20 min 10 s post bubble administration (R20) was used to represent that of the retained-particles only, as signal contribution from any residual freely circulating-particles by this time was negligible due to their low concentrations in the central blood pool (signal intensity in the LV cavity). E.g., 0.1 AU in the LV cavity would contribute only 0.005-0.024 AU in the myocardium assuming a relative myocardial blood volume (% of myocardium that is blood) of ≈5-24%..sup.65-67 R20 was significantly higher in the WT (0.46±0.16 (SEM), range 0.01-1.61, n=12) than KO (0.06±0.03, −0.01-0.24, n=8) animals, p<0.05. R20 correlated strongly with LPS.sub.Time in the WT (r=−0.78, p<0.05, n=12) but not KO mice (r=−0.05, p=0.9, n=8), FIG. 8c. It also correlated strongly with the concentration of Esel mRNA (r=0.81, p<0.005, n=12), FIG. 8d. [0196] (3) TIC-based method. The maximum retained-particle signal intensity in the myocardium. A.sub.r, was significantly higher in the WT (2.3±0.4 (SEM), range 0.8-4.2, n=12) than Esel KO (0.4±0.1, 0-1, n=8) animals, p<0.005. The low A.sub.r values in the KOs suggested minor degrees of non-specific particle retention. A.sub.r correlated strongly with LPS.sub.Time in the WT (r=−0.87, p<0.0005, n=12) but not KO mice (r=−0.08, p=0.86, n=8), FIG. 8a. By converting LPS.sub.Time to the concentration of Esel mRNA using the curve in FIG. 7a, FIG. 8b showed that A.sub.r correlated strongly with the concentration of Esel mRNA (r=0.84, p <0.001, n=12), the latter in the range that was approximately linearly related to the concentration of the cell surface Esel protein, FIG. 7c.

[0197] Acoustic Quantification of Particle Half-Life In Vivo (Using TIC-Based Method)

[0198] The half-life of the freely circulating-particles in vivo was ≈2(mean) 1(SD) min, range 1-5 min for the WT (n=12) and KO (n=8) animals. This was comparable with most commercially available non-targeting microbubbles (range ≈1-3 min), demonstrating that the microparticles produced were of commercial quality or better. The elimination of the retained-particles in the myocardium decreased with increased maximum concentration of retained-particles in the myocardium. The relationship was non-linear and could be empirically fitted to an exponential or sigmoidal function. This resulted in the half-life of the retained-particles being shorter the lower the maximum retained-particle concentration. The in vivo half-life of the acoustically effective retained-particles in the myocardium in these groups of animals was ≈7(mean)±5(SD) min, range 3-18 min in the WT (n=12) compared with 4±4 min, range 1-14 min in the KO animals (n=8).

[0199] Targeting Microparticle Pharmacokinetics and Accumulation Kinetics on the Target Surface

[0200] The accumulation of attached microparticles on the target surface was essentially complete soon after particle-bolus administration. Using the novel TIC-based method, it was discovered that the elimination of the retained and freely-circulating targeting microparticles followed first-order kinetics in vivo.

[0201] These results demonstrate that the targeting microparticles are specific and effective in vivo for highly quantitative real-time ultrasound molecular imaging of one or more organs. The microparticles have favourable characteristics in vivo which include, but are not limited to, being non-toxic, sufficiently stable for continuous and multi-plane imaging with a single-bolus microparticle administration, having favourable kinetics and acoustic response for highly quantitative analysis of the molecular moiety of interest. They have sufficiently high targeting specificity and efficiency to the molecular moiety of interest in vivo, and lack non-specific binding/persistence in tissues not expressing the molecular moiety of interest (except in the liver and spleen which are the usual routes of microparticle elimination in the body). In conclusion, these targeting microparticles are different and superior to the prior art.

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