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99mTc-Hexamethylpropyleneamine oxime pH-sensitive liposomes

99mTc-SpHL

, PhD, , PhD, , PhD, and , PhD.

Author Information

Created: ; Last Update: August 7, 2008.

Chemical name:99mTc-Hexamethylpropyleneamine oxime pH-sensitive liposomes
Abbreviated name:99mTc-SpHL
Synonym:
Agent Category:pH-Sensitive liposomes
Target:Inflamed tissue
Target Category:Uptake
Method of detection:Single-photon emission computed tomography (SPECT); gamma planar imaging
Source of Signal/Contrast:99mTc
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents

Background

[PubMed]

Inflammatory and infectious diseases are a common cause of patient morbidity and mortality despite recent advances in antimicrobial therapy (1, 2). The detection and diagnosis of these lesions is essential for the development of appropriate and timely treatment of the ailment. Scintigraphy plays an important role in the assessment of patients suspected to have these illnesses because this technique is based on the in vivo detection of radiotracer distribution, which is usually based on the physiological characteristics of the tissues. Therefore, this method allows detection of the physiopathological processes during the initial stages and differs from the conventional methods that are based on visualization of anatomic alterations (3-5). Liposomes are nanocarriers that have been widely used for the in vivo delivery of a variety of drugs and molecules to detect or treat different pathological conditions such as cancer or microbial infections, or even used for the delivery of gene therapy (6-8). In addition, liposomes that are pH-sensitive have been developed and used for the preparation and delivery of radiolabeled drugs to detect and treat tissue lesions (9). An acidic environment in the inflamed or infected tissues collapses the liposomes into a non-bilayer structure, which leads to its disruption and the subsequent release of the trapped radioactive marker(s). Thus, these liposomes can be used to enhance the bioavailability of a radiotracer and improve the scintigraphic imaging quality. On the other hand, one limitation of the use of liposomes as diagnostic agents is the aspect of complement activation, which results in liposome clearance by the mononuclear phagocyte system (MPS) and may lead to the development of hypersensitivity in the treated individuals (10, 11). It has been reported that the inclusion of amphipathic poly(ethylene glycols) (PEGs) in the liposome composition can significantly reduce liposome uptake by the MPS, resulting in prolonged liposome circulation (9, 12). It is believed the liposomes accumulate around the tissue cells due to vasodilation and some macrophages may phagocytise the liposomes as a defense mechanism resulting in some inflammation (13). Carmo et al. developed a novel, meta-stable technetium (99mTc) containing hexamethylpropyleneamine oxime (HMPAO) and pH-sensitive liposomes (99mTc-SpHL) and investigated its tissue distribution, ability to detect inflammation, and complement activation in rats (14).

Synthesis

[PubMed]

99mTc-SpHL was prepared as described elsewhere (15). Briefly, a lipid mixture containing dioleoylphosphatidylethanolamine, cholesteryl hemisuccinate, and methoxypoly(ethylene glycol)2000-distearoylphosphatidylethanolamine at a molar ratio (total lipid) of 6.5:3.0:0.5 were transferred as chloroform aliquots of the lipids to a round-bottom flask, and a lipid film was formed by rotary evaporation under reduced pressure. The resulting lipid film was hydrated with HEPES buffer (pH 7.4) containing 50 mM reduced glutathione, and the liposome suspension was then filtered through polycarbonate membranes with a medium pressure extruder. To label HMPAO with 99mTc, 125 µg of the compound (in saline) was incubated with 1,184 MBq (32 mCi) 99mTc-labeled sodium pertechnetate for 5 min at 37°C. Finally, the pH-sensitive liposomes containing glutathione were incubated with freshly prepared 99mTc-HMPAO (37 MBq (1 mCi)/µmol of phospholipids) for 30 minutes at 37°C to yield 99mTc-SpHL (16). Unencapsulated 99mTc-HMPAO was removed with gel filtration on a Sephadex G-25 column with HEPES buffer (pH 7.4) as an eluent. The mean diameter and polydispersity index of the vesicle dispersion were determined to be 123.2 ± 9.0 nm and 0.19 ± 0.02, respectively. The labeling efficiency of the liposomes was 85% as determined with a dose calibrator before and after gel filtration (14).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

A hemolytic assay with rat serum (RS) and antibody-sensitized sheep red blood cells (SRBC) was used to assess the effect liposomes had on functional complement activation in serum (17, 18). First, an SpHL suspension diluted in veronal buffer solution containing MgCl2 and CaCl2 (VBS2+) was incubated with RS for 30 minutes at 37°C (diluted 1:3 in VBS2+). After incubation, ice-cold VBS2+ (300 µl) was added to the mixture, and the mixture was put on ice. Briefly, sheep erythrocytes were sensitized with the use of rabbit anti-SRBC antibodies (antibody-coated erythrocytes (EA)) and suspended at a concentration of 1 × 108 cells/ml in VBS2+. Then, RS exposed to the liposomes (50 µl) was incubated with EA (50 µl) under agitation for 30 minutes at 37°C. The reaction was stopped by addition of ice-cold VBS2+ containing 10 mM of ethylenediaminetetraacetic acid. For control, RS unexposed to liposomes was used. Unlysed sheep erythrocytes were removed with centrifugation, and the amount of hemoglobin released into the supernatant was measured spectrophotometrically at a wavelength of 414 nm. In the absence of lipids, 100% lysis of the SRBCs was observed, indicating no complement consumption. The SpHL was observed to be a poor activator of the complement and was able to induce complement activation only if it contained >8 nmol of phospholipids (14).

Animal Studies

Rodents

[PubMed]

Biodistribution studies of 99mTc-HMPAO and 99mTc-SpHL were performed in Wistar rats bearing inflammation induced by subplantar injection of carrageenan (1%) in the right foot (14). At 0.5, 2, 4, 6, and 8 h (n = 5 animals/time point) after intravenous injection of the respective radiotracers, the animals were anaesthetized for the removal of affected tissues; a γ-scintillation counter was used to determine the 99mTc content. Scintigraphic imaging was also performed at 2, 4, and 8 h after intravenous injection of 99mTc-SpHL. The results of biodistribution studies showed that a significant quantity of 99mTc-SpHL was taken up by the spleen (19.21 ± 2.98% injected dose/g tissue (% ID/g)) at 30 min and was maintained for up to 4 hours after administration. These findings are in agreement with expected results as well as with results observed by other authors using long-circulating liposomes, considering that the spleen is the MPS organ responsible for blood clearance (19, 20). The same uptake pattern was observed after administration of 99mTc-HMPAO alone; however, the level of radioactivity accumulation was 18 times lower than that observed with 99mTc-SpHL. Low radioactivity levels were detected in the liver, lungs, and kidney at all of the time points investigated after the administration of either radiotracer. The uptake of 99mTc-SpHL was significantly higher (P < 0.001) in the inflamed foot than in the respective control at all of the time points investigated. The 99mTc-SpHL levels (% ID/g) were 0.43 ± 0.06 (0.5 h), 0.38 ± 0.05 (2 h), 0.40 ± 0.04 (4 h), 0.34 ± 0.07 (8 h), and 0.24 ± 0.03 (18 h), whereas the 99mTc-HMPAO levels (% ID/g) observed were 0.25 ± 0.02, 0.18 ± 0.02, 0.16 ± 0.02, 0.19 ± 0.02, and 0.15 ± 0.01, at the respective time points (14).

Images obtained 2 h after intravenous administration of 99mTc-SpHL in Wistar rats that presented inflammation sites in the right foot showed an accumulation of radioactivity in the inflamed region (14). The radioactivity level in this region could be observed up to 8 h after injection of the liposomal radiotracer. The target/non-target tissue ratio was 5.49 ± 0.74 at 2 h after administration, and the ratio increased over time (10.39 ± 1.33 at 8 h). This indicated a preferential retention of 99mTc-SpHL in the inflammation site compared to the non-inflamed contralateral foot.

With observations from this study, the investigators concluded that 99mTc-SpHL accumulates specifically in the inflamed lesions but is rapidly removed from non-target tissues to allow for the visualization of the lesions shortly after administration of the radiotracer.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimers]

References

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2.
Cirino G. Multiple controls in inflammation. Extracellular and intracellular phospholipase A2, inducible and constitutive cyclooxygenase, and inducible nitric oxide synthase. Biochem Pharmacol. 1998;55(2):105–11. [PubMed: 9448732]
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    10.
    Szebeni J. Complement activation-related pseudoallergy caused by liposomes, micellar carriers of intravenous drugs, and radiocontrast agents. Crit Rev Ther Drug Carrier Syst. 2001;18(6):567–606. [PubMed: 11789676]
    11.
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  • 14.
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    This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.

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