3.1 Liposome diameter, zeta potential, Lipo-PEG-Peptide (LPP) incorporation, and in vitro binding
The LPP (structure shown in ), radiolabeled lipid ([18F]FDP), phospholipid (DPPC), and pegylated phospholipid (DSPE-PEG2000) were combined to produce a particle (, with formulations and notation detailed in caption), with a polymer brush layer of 2000 molecular weight (MW) and a ligand that was either exposed (m=3 in ) or buried (m=1). Initial in vitro studies indicated that the formulation of CRPPR-3:DSPE-PEG2000:DPPC = 6%:6%:88% (mol/mol) produced effective targeting, therefore this formulation was employed in in vitro studies and as the baseline formulation in in vivo studies. In vivo results with the CRPPR-3:DSPE-PEG2000:DPPC = 6%:6%:88% were compared with matched total PEG concentration (12%), LPP concentration (6%), DSPE-PEG2000 concentration (6%), or LPP:DSPE-PEG2000 ratio (1:1), while other parameters were varied ().
Liposome diameter, zeta potential, and lipoPEGpeptide incorporation as a function of formulation*
Using flow cytometry (), both endothelial and melanoma cells incubated in culture with calcein-containing particles targeted by CRPPR-3 or CPPRR-3 showed a significantly higher fluorescence intensity than cells incubated with particles without the lipopeptide (p<0.05). Endothelial cells incubated with particles containing CRRPP-3 demonstrated a lower fluorescence intensity than those incubated with particles containing CRPPR-3 or CPPRR-3.
3.2 PET images
Ninety-minute accumulative PET images () acquired with the [18F]FDP and CRPPR-3 incorporated into the liposomal vectors () demonstrate the high level of the radiotracer within the heart, a lower density within the liver, and a low concentration within the spleen and bladder. Images acquired with the shorter PEG length LPP (CRPPR-1) loaded onto an identical vehicle demonstrate that radioactivity has accumulated within the liver and bladder at 90 minutes and a low level of activity is present in the heart (). Clearance of the radiotracer occurs through the bladder, after metabolism in the liver separates the fatty acid chains (each with a molecular weight of 256) from the head group containing the isotope (with a molecular weight of 94). By comparison, images acquired with the identical vehicle, without a peptide attached to the liposome, demonstrate that the radioisotope is primarily circulating within the blood volume throughout the ninety-minute scan, visualized in the heart chamber and carotid vessels ().
(a–i) 90-minute accumulative PET images acquired after injection of radiolabeled liposomes from coronal (a–c), sagittal (d–f) and transverse views (g–i) with LPPs CRPPR-3 (a,d,g), CRPPR-1 (b,e,h), and NON (c,f,i).
Autoradiography confirmed that the radiotracer was present throughout the atria and both ventricles (), although the highest counts were observed within the thick ventricular walls. Confocal microscopy confirmed that CRPPR-targeted liposomes bind to blood vessel walls within the heart ().
Figure 3 High resolution autoradiography and optical imaging of the heart after injection of targeted and control particles. (a–d) Autoradiography images acquired from 60 µm tissue slices 90 minutes after injection of CRPPR-3 liposomes. (e) Anatomic (more ...)
3.3 Effect of peptide and surface architecture on biodistribution at 90 minutes
Well counts obtained from harvested tissues at the ninety-minute time point quantify the differences in biodistribution produced by the peptide (), the length of the PEG spacer between the fatty acid and CRPPR peptide portions of the LPP (), and the molar fraction of the CRPPR-3 incorporated within the vehicle (). When injected on a vehicle containing CRPPR-3, the concentration of the tracer was significantly higher within the heart than other organs (p<0.001), with a target to skeletal muscle ratio as high as 100 in individual animals (averaging 32) and a mean heart concentration of 44 ± 9% injected dose per gram of tissue (ID/g). For CRPPR-3, activity within the liver and spleen was significantly lower than the heart at 22 ± 9 and 12 ± 4% ID/g (p<0.001). Other arginine-rich peptidyl liposomes bound to the heart () at levels of 39 ± 13, 26 ± 13, 17 ± 5% ID/g for CPPRR-3, CRRRR-3, and CRRPP-3, respectively, with target to skeletal muscle ratios of 32, 23 and 19. Injection of vehicles with the cyclized RGD peptide and an otherwise identical liposome surface architecture did not produce radioactivity above the baseline (no–peptide) case. The cyclized RGD and no-peptide controls also resulted in significantly lower activity levels within the liver and urine at 90 minutes as compared with arginine-rich peptidyl vehicles (p<0.05).
Figure 4 Well counts (%ID/g) obtained 90 minutes after injection. (a), (b) and (c), with different LPP; (d), with varied ratios of CRPPR-3:DSPE-PEG2000. Significance of the accumulation of particles targeted with CRPPR-3:DSPE-PEG2000 6%:6% tested against other (more ...)
When the PEG spacer length supporting the peptide was decreased from 3600 to 1200 MW within a surrounding brush layer of DSPE-PEG2000, such that the peptide was shielded by the brush layer, binding of the isotope-containing particle decreased ~10 fold (). For particles targeted with CRPPR-1, the isotope concentration within the urine at 90 minutes was greatly increased (p<0.01), demonstrating the rapid clearance of the tracer. For particles targeted with CRPPR-2, isotope accumulation within the heart was in all cases less than particles containing CRPPR-3 (p<0.05), and greater than particles containing CRPPR-1 (p<0.001).
Radioactivity detected within the heart increased with increasing CRPPR-3 content from 2 to 6%, with the molar percent of DSPE-PEG2000 held constant at 6% (p<0.001, ). Neither LPP incorporation or resulting radioactivity increased further as the CRPPR-3 content was increased to 10% (with 2% DSPE-PEG2000), although we note that the 10% CRPPR-3 particles were difficult to extrude and had a higher mean diameter of 192 ± 89nm (). For particles with 2% CRPPR-3, increasing the molar percentage of DSPE-PEG2000 from 6 to 10% increased the percentage circulating within the blood (p<0.001) at 90 minutes but further decreased the activity within the heart (p<0.01) (data not shown).
3.4 Real-time pharmacokinetics
Dynamic PET analysis provides the opportunity to evaluate the rate of accumulation of the isotope at the target site and to detect accumulation in unexpected targets in real time (Supplemental Video 1
). Accumulation of radiolabeled-particles containing CRPPR-3 was very rapid (tens of seconds) within the heart (). Particles containing CRPPR-1 cleared rapidly from the heart, with activity significantly below CRPPR-3 from 40 seconds after the start of the injection () (p<0.01). Since the heart region of interest (ROI) evaluated with microPET also includes circulating blood within the heart, the heart TAC resulting from the injection of particles containing CRPPR-1 (which did not bind but had similar blood pharmacokinetics to CRPPR-3) was subtracted from the CRPPR-3 TAC. The resulting plot () indicates that the bound activity within the heart increases rapidly over an interval less than 100 seconds, and continues to increase at a slower rate over the duration of the scan. When fit to a single exponential, a time constant of ~30 seconds for accumulation of activity for particles containing CRPPR-3 was estimated based on .
Figure 5 Time activity curves (TACs) and Logan Plot from dynamic PET analysis of various LPP liposomes. (a)–(b) TACs for heart muscle, (c) difference between CRPPR-3 and CRPPR-1 liposomes in TAC from heart muscle. Subtraction of non-binding CRPPR-1 particles (more ...)
The volume of distribution of different tracers in the myocardium was calculated using a Logan plot [28
]. The volume of distribution of particles containing CRPPR-3 (for example ) is significantly larger than CRPPR-1 () (p<0.001), indicating higher binding avidity. Note that the estimated values are conservative, particularly for CRPPR-3, as the effects of possible metabolites in blood and the contribution of the blood activity in the myocardial region were not accounted for. Following correction for circulating metabolites, the true volume of distribution of particles containing CRPPR-3 can be significantly greater than four.
Gross differences between PET images were visible in the blood clearance rate of particles depending on the attached peptide (). Estimates of radioactivity within the blood pool over time were fit with a biphasic clearance curve (1)
, with the constants as shown in . Without an LPP and with 12% DSPE-PEG2000 (NON in ), the particle was long circulating, with an α value of 104
seconds. Without an LPP and with 6% DSPE-PEG2000, the particle is similarly long circulating (data not shown). Particles coated with RGD-3 were long circulating as indicated by α and β values of 103
seconds, respectively. The presence of CRPPR, CPPRR, CRRPP, or CRRRR on the liposome substantially reduced the circulation time (even with the peptide shielded by a longer brush layer) with α values of ~102
Circulation time constants for [18F] in the blood pool and peak concentrations and accumulation time constants for organs as assessed from PET TACs
3.5 RES recognition and clearance
For particles containing the arginine-rich linear peptide, accumulation in the liver is very rapid (570 seconds or less in all cases) ( and ). Alternatively, particles containing RGD-3 reach a greater peak concentration within the spleen as compared with the liver (p<0.05), with the peaks observed at 3300 and 1050 seconds for spleen and liver, respectively.
With CRPPR-1-targeted particles, clearance of the radioisotope from the liver (and accumulation in the bladder) was more rapid than observed with the longer PEG spacer (CRPPR-3). Particles targeted with CRRRR-3 also accumulated more rapidly in the bladder than CRPPR-3 ().
For particles containing RGD-3, the concentration within liver, spleen and bladder () indicate that a fraction was metabolized through the liver (concentration peaking at 1050 seconds) and cleared through the urine. At the 90 minute time point, accumulation of activity within the liver was not significantly different than the no-peptide control (p=0.17).
3.6 RES inhibitors
In order to increase the accumulation at the target site, several methods for the reduction of liver and spleen uptake were evaluated, including the pre-administration of polyinosinic acid, blank liposomes, clodronate liposomes, or free CRPPR peptide. Pre-administration of 12 milligram per kilogram of body weight (mg/kg) of blank liposomes did not significantly change the biodistribution of the targeted liposomes (p=0.10, data not shown).
When clodronate liposomes were administered 24 hours before particles containing CRPPR-3, the circulation time of the particles and binding of the particles to the heart increased at the early time points (before 500sec), each as compared with matched controls not receiving clodronate (p<0.001, ). However, radioactivity in the heart decreased by 10% over the 90-minute scan (not observed in the absence of clodronate), and activity simultaneously increased within the spleen ().
Figure 6 TACs from dynamic PET analysis for (a) heart muscle (b) blood pool (c) liver (d) spleen (e) bladder after injection of CRPPR-3 liposomes and various inhibitors. Abbreviations: CRPPR-3: no inhibitors; CLD: clodronate liposomes injected 24 hours in advance; (more ...)
Early liver uptake (before 1000 seconds) was significantly lower than observed without clodronate (p<0.05) (). Liver activity does not decrease over the scan (ending higher than without clodronate) (), indicating that the probe is not metabolized and cleared, and the accumulation of activity within the urine at the 90-minute time point was significantly decreased (p<0.05) (data not shown).
Pre-administration of polyinosinic acid (0.4mg/kg) decreased accumulation within the heart by 41% at the 90 minute time point (p<0.05), and this decrease was significant from the six-minute time point forward (p<0.01, ). Pre-administration of the free CRPPR peptide (25mg/kg) decreased the accumulation within the heart (p<0.05, ) by ~19% at the 90 minute time point (p<0.05), showing the specificity of the targeting.