In vivo evaluation of PEGylated ⁶⁴Cu-liposomes with theranostic and radiotherapeutic potential using micro PET/CT

Abstract

Purpose

The objective of this study was to evaluate the potential of PEGylated ⁶⁴Cu-liposomes in clinical diagnostic positron emission tomography (PET) imaging and PEGylated ¹⁷⁷Lu-liposomes in internal tumor radiotherapy through in vivo characterization and dosimetric analysis in a human xenograft mouse model.

Methods

Liposomes with 5 and 10 mol% PEG were characterized with respect to size, charge, and ⁶⁴Cu- and ¹⁷⁷Lu-loading efficiency. The tumor imaging potential of ⁶⁴Cu-loaded liposomes was evaluated in terms of in vivo biodistribution, tumor accumulation and tumor-to-muscle (T/M) ratios, using PET imaging. The potential of PEGylated liposomes for diagnostic and therapeutic applications was further evaluated through dosimetry analysis using OLINDA/EXM software. The ⁶⁴Cu-liposomes were used as biological surrogates to estimate the organ and tumor kinetics of ¹⁷⁷Lu-liposomes.

Results

High remote loading efficiency (>95 %) was obtained for both ⁶⁴Cu and ¹⁷⁷Lu radionuclides with PEGylated liposomes, and essentially no leakage of the encapsulated radionuclide was observed upon storage and after serum incubation for 24 h at 37 °C. The 10 mol% PEG liposomes showed higher tumor accumulation (6.2 ± 0.2 %ID/g) than the 5 mol% PEG liposomes, as evaluated by PET imaging. The dosimetry analysis of the ⁶⁴Cu-liposomes estimated an acceptable total effective dose of 3.3·10⁻² mSv/MBq for diagnostic imaging in patients. A high absorbed tumor dose (114 mGy/MBq) was estimated for the potential radiotherapeutic ¹⁷⁷Lu-liposomes.

Conclusion

The overall preclinical profile of PEGylated ⁶⁴Cu-liposomes showed high potential as a new PET theranostic tracer for imaging in humans. Dosimetry results predicted that initial administered activity of 200 MBq of ⁶⁴Cu-liposomes should be acceptable in patients. Work is in progress to validate the utility of PEGylated ⁶⁴Cu-liposomes in a clinical research programme. The high absorbed tumor dose (114 mGy/MBq) estimated for ¹⁷⁷Lu-liposomes and the preliminary dosimetric studies justify further therapeutic and dosimetry investigation of ¹⁷⁷Lu-liposomes in animals before potential testing in man.

Appendices

Appendix A: Tables of absorbed radiation doses

Table 3 Absorbed radiation doses of ⁶⁴Cu-liposomes

Table 4 Absorbed radiation doses of ¹⁷⁷Lu-liposomes calculated from the ⁶⁴Cu-liposome distribution

Appendix B: Equations 1–6

The percentage of injected dose in blood (%ID_blood) was calculated using:

$$ \%I{D}_{blood}=\frac{A_b\cdot 7.5\%\cdot m}{D} $$

(1)

where A _b is the decay-corrected blood activity concentration at time t. The animal’s blood volume was calculated as 7.5 % of its body weight, m. D is the injected activity.

The percentage of injected dose in blood (%ID_blood) between 1 and 48 h was fitted to the mono-exponential equation:

$$ \%I{D}_{blood}(t)=B\cdot {e}^{-\beta t} $$

(2)

where β is the first-order disposition rate constant or the elimination rate constant, and B represents the fraction of the injected dose which is cleared from the blood at the disposition rate β. The fraction of the dose cleared initially can be estimated by 100 % -B. The terminal half-life (T _½β ) of the liposomes was determined using:

$$ {T}_{1/2\beta }=\frac{ \ln \kern.15em 2}{\upbeta} $$

(3)

The available data did not permit accurate estimation of an initial half-life (T _½α ).

Accumulation in tumor and organs

Liposomal concentrations within each source organ as a function of time were determined from an ROI placed over the entire volume of the organ. The liposomal accumulation in the different organs was expressed as percentage of injected dose per gram (%ID/g) by using:

$$ \% ID/g=\frac{A}{D\cdot \rho } $$

(4)

where A is the decay-corrected activity concentration in the tumor and normal organs, and ρ is the organ density and is assumed to be 1 g/cm³ for all organs and tumors.

The liposomal accumulation in the tumor was further parameterized by the standardized uptake value (SUV) and the tumor-to-muscle (T/M) ratio. The SUV values were calculated using:

$$ SUV=\frac{A\cdot m}{D\cdot \rho } $$

(5)

where m is the animal's body weight and A is the decay-corrected activity concentration in the tumor. The PET data was not corrected for attenuation, which would give 5–15 % higher ⁶⁴Cu concentrations than the values provided in this study, depending on the tissue in question.

For the ⁶⁴Cu-liposome dosimetry study, activity concentration data, which were not corrected for ⁶⁴Cu radioactive decay (Â), were used to construct activity concentration-time curves for each source organ. For dose calculations the the program OLINDA/EXM [27] were used. The residence time (T _R ) from each source organ was used as input, and was calculated as:

$$ {T}_R=\frac{V_{organ}}{D}\cdot {\displaystyle {\int}_0^{\infty}\widehat{A}\ dt} $$

(6)

where  is the activity concentration in the organ as a function of time, and V _organ is the volume of the source organ. Integration was carried out using the trapezoidal method to obtain the area up to the last measured activity concentration ( ^∗). An estimate of the long-term tail of the activity concentration-time curves for the different organs was made by fitting an exponential function ( ~ A ⋅ e ^− βt) to the two last measured points. The area beyond  ^∗ was then estimated by  ^∗/β. Since steady-state condition for the liposomal concentration within the measured tumor tissue was observed after 24 h (data not shown), the long-term tail of the activity concentration-time curves was assumed to be governed only by the radionuclide decay. The area beyond  ^∗ was thus estimated by ${\widehat{A}}^{\ast}\cdot \frac{T_{1/2}}{ln2}$, where T _½ is the half-life of the radionuclide.

In the OLINDA/EXM program, activity not assigned to a specific organ must be assigned to the remainder-of-body category. In our calculation, this was estimated as the full injected dose activity received by the body excluding the source organ doses. A separate T _R was assigned to the remainder-of-body activity concentration-time curve.

For the ¹⁷⁷Lu-liposome dosimetry study, ⁶⁴Cu-liposomes were used as biological surrogates to study the biodistribution and estimate radiation dosimetry of ¹⁷⁷Lu-liposomes. Thus, it was assumed that the ¹⁷⁷Lu-liposomes follow virtually the same biodistribution and pharmacokinetics as the ⁶⁴Cu-liposomes due to the encapsulation of the radionuclides inside the liposomes prohibiting the exchange of the radionuclides with the biological environment. ⁶⁴Cu-liposome activity data for each source organ corrected for ⁶⁴Cu radioactive decay were multiplied by the physical decay of ¹⁷⁷Lu (${e}^{-\frac{ln2}{T_{1/2}}}$) to obtain estimates for the ¹⁷⁷Lu-liposome activity concentration in each source organ as a function of time. The T _R from each source organ was calculated via equation S6.