Cholesterol enhances the delivery of liposome-encapsulated gallium-67 to tumors

The effect of incorporation of cholesterol (CH) into liposome membranes on the delivery of 67Ga encapsulated in liposomes to tumors was studied. The changes of the blood clearance of liposomes, liposome stability in serum and liposome uptake by the liver and spleen were also examined. Liposomes were prepared from distearoylphosphatidylcholine with various amounts of CH. It became clear that large amounts of CH (above 33 mol%) dramatically enhanced liposomal delivery of 67Ga to sarcoma 180 solid tumor in mice. Large amounts of CH increased the liposome stability in serum and decreased liposome uptake by the liver and spleen after intravenous injection, thus prolonging the blood clearance of liposomes. These observations suggested that the large delivery of 67Ga by CH-rich liposomes resulted from the extended retention and increased amount of liposomes in the circulation, caused by the incorporation of large amounts of CH. Small amounts of CH decreased liposome stability and hastened blood clearance, but had little effect on 67Ga delivery to the tumor. CH-rich liposomes showed high tumor uptake, with high tumor to blood and tumor to tissue ratios of 67Ga. It is anticipated that 67Ga-carrying liposomes will be an excellent tumor imaging agent for clinical use, provided that a correct choice of CH content is made.     

Cholesterol enhances the delivery of liposome-encapsulated gallium-67 to tumors

The effect of incorporation of cholesterol (CH) into liposome membranes on the delivery of ⁶⁷Ga encapsulated in liposomes to tumors was studied. The changes of the blood clearance of liposomes, liposome, stability in serum and liposome uptake by the liver and spleen were also examined. Liposomes were prepared from distearoylphosphatidylcholine with various amounts of CH. It became clear that large amounts of CH (above 33 mol%) dramatically enhanced liposomal delivery of ⁶⁷Ga to sarcoma 180 solid tumor in mice. Large amounts of CH increased the liposome stability in serum and decreased liposome uptake by the liver and spleen after intravenous injection, thus prolonging the blood clearance of liposomes. These observations suggested that the large delivery of ⁶⁷Ga by CH-rich liposomes resulted from the extended retention and increased amount of liposomes in the circulation, caused by the incorporation of large amounts of CH. Small amounts of CH decreased liposome stability and hastened blood clearance, but had little effect on ⁶⁷Ga delivery to the tumor. CH-rich liposomes showed high tumor uptake, with high tumor to blood and tumor to tissue ratios, of ⁶⁷Ga. It is anticipated that ⁶⁷Ga-carrying liposomes will be an excellent tumor imaging agent for clinical use, provided that a correct choice of CH content is made.     

Tumor uptake of 67Ga-carrying liposomes

The in vivo distribution, excretion, and tumor localization of liposome-encapsulated 67Ga in normal and Ehrlich tumor (solid form)-bearing mice were studied. In normal mice, multilamellar vesicles (MLVs) were taken up mainly by the liver and spleen, whereas small unilamellar vesicles (SUVs) exhibited a broader tissue distribution. When 67Ga was encapsulated in MLVs or SUVs, the excretion of the radiotracer in the urine and feces was less than that observed for free tracer at 72 h after i.v. administration. In tumor-bearing mice, SUVs were found to accumulate preferentially in tumors. The tumor uptake of neutral, positive, and negative SUVs was 10%-13% of the administered dose per gram of tumor tissue at 24 h after their injection. These values were about three times higher than those found for free 67Ga-nitrilotriacetic acid (67Ga-NTA) or 67Ga-citrate. Significant differences in tumor uptake due to different surface charges of liposomes were not observed. Enhanced tumor-to-blood and tumor-to-muscle ratios were also observed at 24 h after injection. These results suggest that 67Ga-carrying liposomes may be a useful for tumor imaging.     

Tumor uptake of 67Ga-carrying liposomes

The in vivo distribution, excretion, and tumor localization of liposome-encapsulated ⁶⁷Ga in normal and Ehrlich tumor (solid form)-bearing mice were studied. In normal mice, multilamellar vesicles (MLVs) were taken up mainly by the liver and spleen, whereas small unilamellar vesicles (SUVs) exhibited a broader tissue distribution. When ⁶⁷Ga was encapsulated in MLVs or SUVs, the excretion of the radiotracer in the urine and feces was less than that observed for free tracer at 72 h after i.v. administration. In tumor-bearing mice, SUVs were found to accumulate preferentially in tumors. The tumor uptake of neutral, positive, and negative SUVs was 10%–13% of the administered dose per gram of tumor tissue at 24 h after their injection. These values were about three times higher than those found for free ⁶⁷Ga-nitrilotriacetic acid (⁶⁷Ga-NTA) or ⁶⁷Ga-citrate. Significant differences in tumor uptake due to different surface charges of liposomes were not observed. Enhanced tumor-to-blood and tumor-to-muscle ratios were also observed at 24 h after injection. These results suggest that ⁶⁷Ga-carrying liposomes may be a useful for tumor imaging.     

Usefulness of<Superscript>67</Superscript>Ga scintigraphy in extranodal malignant lymphoma patients

OBJECTIVE: 67Ga scintigraphy has a well-documented role in nodal lymphoma for both disease staging and assessment of treatment response. The objective of the present study was to examine the role of 67Ga scintigraphy in diagnosis and assessment of treatment response, in patients with extranodal malignant lymphoma. METHODS: Seventy-one patients with extranodal malignant lymphoma were studied. Whole body scans in all and SPECT scans in some selected patients were performed 72 hours after injection of 67Ga-citrate. The influence of tumor site, histological classification and tumor size on 67Ga scintigraphy sensitivity was analyzed. Twenty-one of the seventy-one patients also had a second 67Ga scintigraphy to assess response to treatment. RESULTS: The overall 67Ga scintigraphy sensitivity was 83.1% (59/71). The sensitivity was low in patients whose extranodal lymphoma occurred in skin (0/3) and urinary bladder (0/1), as compared to other tumor sites. According to the histological classification of the lesion, the sensitivity was lower in low-grade than in intermediate and high-grade lymphoma. According to the tumor size, the sensitivity was low in lesions less than 2 cm in diameter than those more than 2 cm in diameter. The results changed from positive to negative accumulation in 20 (95.2%) of the 21 patients who had 67Ga scintigraphy to assess the response to treatment. These 20 patients showed a good clinical course. CONCLUSIONS: Although 67Ga scintigraphy did not show positive accumulation in patients with skin and urinary bladder lymphoma, it was helpful to confirm the diagnosis and to evaluate the therapeutic effect in most patients with extranodal malignant lymphoma.     

Renal gallium accumulation in mice with acute immune complex glomerulonephritis

67Ga uptake and heparan sulfate (HS) content were investigated during the recovery of mouse kidney from acute immune complex glomerulonephritis induced by daily injections of bovine serum, and the binding of 67Ga to glomerular basement membrane (GBM) was studied in vitro. The results were as follows. 67Ga uptake in the kidney increased after the start of bovine serum injection, and peaked on the 20th day. The uronic acid content in 1.2 M NaCl-soluble fraction (which contained predominantly HS) and the hydroxyproline content (an index of collagen) were increased at the 10th day, reaching a maximum at the 20th day. This pattern of HS content was essentially the same as that of 67Ga accumulation in the kidney. Urinary protein and gamma-GTP activity peaked at the 5th day, and these patterns were different from that of 67Ga uptake. 67Ga binding to GBM was significantly inhibited by treatments with HS-degrading enzyme (heparitinase), nitrous acid, trypsin or papain. However, the binding to GBM was unaffected by treatment with chondroitinase ABC. These results provide further evidence that the 67Ga-binding substance in tumor tissues and inflammatory lesions is probably HS.     

Enhanced tumor MR imaging with gadolinium-loaded polychelating polymer-containing tumor-targeted liposomes

PURPOSE: To significantly enhance tumor MR imaging by using a contrast agent combining three components -- a long-circulating liposome, liposomal membrane-incorporated polychelating amphiphilic polymer heavily loaded with gadolinium, and cancer-specific monoclonal antibody 2C5 attached to the liposome surface. MATERIALS AND METHODS: Tumor-bearing animals were imaged prior and 4, 24, and 48 hours after i.v. injection of 2C5-modified and unmodified Gd-PAP-containing PEGylated liposomes. The faster and more specific accumulation of the novel contrast nanoparticles in tumors was also confirmed by 3D angiograms and by direct visualization of Gd-immunoliposomes in tumor sections by confocal microscopy. RESULTS: 2C5-modified Gd-PAP-containing PEGylated liposomes allowed for fast and specific tumor imaging as early as 4 hours postinjection. T1 inversion recovery maps demonstrated a significant increase in tumor-associated R1 in animals injected with antibody-modified Gd-loaded liposomes 4 hours postinjection, followed by a gradual decrease consistent with clearance of the agent from the tumor region. In control animals injected with antibody-free liposomes the corresponding R1 values at all investigated timepoints were significantly smaller. CONCLUSION: The results support the feasibility of using such multifunctional nanoparticular liposome-based agents simultaneously providing prolonged circulation, heavy Gd load, and specific cancer cell recognition as a superior contrast for MR tumor imaging.     

Diagnosis of thyroid lymphoma and follow-up evaluation using Ga-67 scintigraphy

A strong association between malignant lymphoma and Hashimoto's thyroiditis has frequently been reported. However, it is difficult to detect the lymphomatous transformation of Hashimoto's thyroiditis in the early stage. The purpose of the present study was to examine the usefulness of 67Ga scintigraphy in the diagnosis and evaluation of the therapeutic effects during follow-up, in patients with a suspected diagnosis of primary thyroid lymphoma. Twenty-five patients who were suspected of having primary thyroid lymphoma and had undergone 67Ga scintigraphy were studied. 67Ga planar scintigraphy was performed 72 hours after injection of 67Ga-citrate. The degree and pattern of 67Ga accumulation were graded visually. Histopathology on biopsy examination revealed thyroid lymphoma in 17 and Hashimoto's thyroiditis in 8 patients. Abnormal accumulation of 67Ga in the thyroid was seen in all of the 17 thyroid lymphoma cases with additional mediastinal and abdominal involvement in one. Fifteen of 17 thyroid lymphoma patients also underwent 67Ga scintigraphy 2-4 weeks after chemotherapy and/or radiotherapy. All 15 patients showed diminishing 67Ga accumulation and a good clinical course. In one patient with local recurrence, abnormal accumulation could be depicted by follow-up scintigraphy. However, diffuse or enlarged accumulation in the thyroid was seen in all of the 8 Hashimoto's thyroiditis cases. The degree of abnormal accumulation in the thyroid in clinically active phase thyroiditis was more intense than that in the chronic phase thyroiditis. 67Ga scintigraphy was helpful to confirm the diagnosis of thyroid lymphoma and to evaluate the therapeutic effects during follow-up. However, 67Ga scintigraphy may not always distinguish thyroid lymphoma from Hashimoto's thyroiditis, especially the active phase of the disease.     

In vitro and in vivo characterization of 67Ga(3+) complexes with cis,cis-1,3,5-triamino-cyclohexane-N,N',N"-triacetic acid derivatives

The aim of this study was to investigate the in vitro and in vivo performance of a 67Ga complex with cis,cis-1,3,5-triaminocyclohexane-N,N',N"-triacetic acid (tachta) as a potential ligand for use as a Ga(III) radiopharmaceutical for PET imaging. The radiolabeling procedure, electrophoretic properties, lipophilicity, acid stability, human serum stability and biodistribution in mice of 67Ga(tachta) were investigated. The 67Ga(tachta) complex forms at 10(-3) M tachta concentration at 40 degrees C in 100% yield; it is neutral, non-lipophilic, 90% stable at pH = 4 and 5 and 100% stable at pH = 6, for at least 8 d. Serum stability experiments demonstrated that at 5 hr 67Ga(tachta) exists in serum as a free complex. At 24 hr, 30% of 67Ga(tachta) is reversibly bound to transferrin-albumin fraction of serum, and that this percentage remains unchanged for a period of 4 d. Biodistribution in mice showed that 67Ga(tachta) rapidly clears via the kidneys from the body with less than 10% of injected activity left in the body at 3 hours and only 6% remaining after 24 hr. The complex also cleared rapidly from all of the major organs, with bone showing some slightly increased (1.15% ID/g) 24 hr accumulation, in comparison with the 3 hr time point. Based upon these data, 67Ga(tachta) may be considered as a candidate for developing new Ga(III) radiopharmaceuticals for PET.     

Mechanism of gallium-67 accumulation in tumors

Neoplasms are characterized by increased perfusion, increased permeability of their capillary beds to macromolecules, and a delay in new lymphatic vessel growth. These lead to the increased entry and residency time of macromolecules in the interstitial space of tumors. Multiple factors contribute to the localization of 67Ga in tumors. Adequate blood supply is essential; at areas with no blood supply such as the necrotic center of a large tumor, there is no 67Ga accumulation. Gallium-67, mainly in the form of transferrin-67Ga complex, is delivered to the tumor through capillaries with increased permeability. In tumors, some 67Ga is taken up by tumor cells; some may also be taken up by inflammatory cells when they are present. Gallium-67 binding proteins, such as lactoferrin or ferritin, may also contribute to the accumulation and retention of 67Ga in tumors; however, their roles are less clear. The intensity of these various factors determine their relative contribution and the degree of 67Ga accumulation in tumors.     

67Ga accumulation in inflammatory lesion and its mechanism: comparison with malignant tumor

The accumulation of 67Ga in inflammatory lesions increased with time after injection of turpentine oil and reached a plateau 5 days later. At that time the uptake in the lesions was larger than any other tissue, after ten days the lesion uptake decreased. In experiments using rats which had been kept for 5 days after subcutaneous injection of turpentine oil, the accumulation of 67Ga in inflammatory lesions increased with time until six days after administration of 67Ga-citrate. It is clear from this study that 67Ga is avidly accumulated in areas where the subcutaneous tissue is infiltrated with neutrophils and macrophages, that it is not accumulated at the sites in which neutrophils are crowded, that nuclear material, mitochondria, lysosomes and microsomes do not play a major role in 67Ga accumulation in the lesion and that the main binding acid mucopolysaccharide in the lesion is a acid mucopolysaccharide which is none of the following: keratan sulfate, heparan sulfate, heparin, or chondroitin sulfate A, B or C. It is presumed that the main 67Ga binding acid mucopolysaccharide is keratan polysulfate (or other oversulfated acid mucopolysaccharides).     

In vivo monitoring of tissue pharmacokinetics of liposome/drug using MRI: illustration of targeted delivery.

The purpose of this study was to determine if MnSO(4)/doxorubicin (DOX) loaded liposomes could be used for in vivo monitoring of liposome concentration distribution and drug release using MRI. In vitro results show that T(1) shortening correlates with MnSO(4) concentration. Using a temperature-sensitive liposome formulation, it was found that MnSO(4) release significantly shortened T(1). This feature, therefore, suggests that content release can also be measured with these MnSO(4)-loaded liposomes. The feasibility of monitoring this drug delivery and release-imaging agent was shown in a murine tumor model. Upon tumor heating, nonthermally sensitive liposomes selectively but heterogeneously accumulated in the tumor region. The thermally sensitive liposomes showed a clear pattern of accumulation at the periphery of the tumor, concordant with the release temperature of this formulation (39-40 degrees C). This liposome contrast agent has potential for use with hyperthermia by providing individualized monitoring of tissue drug concentration distribution during or after treatment. This would allow for: 1) modification of treatment variables to improve the uniformity of drug delivery, and 2) provide a means to select patients most likely to benefit from this liposomal drug treatment. Additionally, the drug-loading method used for this liposome is applicable to a wide range of drugs, thereby broadening its applicability. The method is also applicable to other liposomal formulations with triggered release mechanisms.     

The mechanism of 67Ga uptake in animal and human tumours

The subcellular distribution of ⁶⁷Ga has been studied by differential centrifugation in 3 transplantable mouse tumours, 3 transplantable rat tumours, 1 dog tumour, 3 human tumour xenografts and 2 human tumours in situ at various times after injection of the citrate complex. From 24 h post injection the nuclide was located predominantly in lysosomal structures in all the tumours studied. Studies in two murine tumours showed marked differences in the rate of lysosomal accumulation of ⁶⁷Ga. In the ADJ/PC6 plasmacytoma lysosomal uptake of ⁶⁷Ga had reached a plateau within 15 min while in the S180 tumour lysosomal accumulation of the nuclide occurred over the first 24 h. Normal mouse liver showed a similar pattern to this latter tumour. It is postulated that these variations in the rate of lysosomal accumulation of ⁶⁷Ga reflect differences in the permeability of the lysosomal membrane. While large amounts of ⁶⁷Ga were found in the crude nuclear fraction of some tumours this was attributed to unbroken cells as studies with purified nuclei from 7 different tumours indicated that between 2 and 14% of the total tumour ⁶⁷Ga was associated with the nuclei.     

泛影葡胺脂质体的制备及其肝脏靶向增强效应观察

目的：探讨泛影葡胺脂质体对肝脏靶向增强的可能。材料和方法：采用乙醚注射法（胆固醇与卵磷脂摩尔比１：１）制备包裹泛影葡胺的大单层脂质体;用ＣＴ观察其对大鼠肝炎型假瘤（ｎ＝４）的靶向增强效果。结果：（１）所制备的装载泛影葡胺脂质体粒径为０．８２±０．３２μｍ,面积为８．４±４．０μｍ２,体积为６．９±１．３μｍ３。造影剂包裹率为１０．１％。体外稳定试验表明泛影葡胺质体放置３ｄ时,含碘量仍保持９３．８％,７ｄ后造影剂大量渗漏,含碘量降至４１．８％。（２）泛影葡胺脂质体能靶向性分布于肝脏,使正常组织呈渐进式增强,３０ｍｉｎ达到高峰,以后逐渐下降,１２ｈ后恢复至基础水平。肝炎性假瘤灶在给药后３０ｍｉｎ显示最佳。结论：泛影葡胺脂质体能有效提高肝脏增强水平,对提高肝脏病变的显示有重要作用。     

Accumulation of indium-111-labeled neutrophils and gallium-67 citrate in rabbit abscesses

A rabbit abscess model was developed to study the effect of abscess age on the accumulation of indium-111-labeled neutrophils ([111In]N) versus gallium-67 citrate (67Ga). Abscesses 1-2 hr, 6-8 hr, 24 hr, and 7 days old were induced by subcutaneous injection of autoclaved colon contents prior to i.v. administration of either [111In]N or 67Ga. Radioactivity in the abscesses was determined 48 hr postinjection. Accumulation of [111In]N was inversely proportional to abscess age. Seven-day-old abscesses were inconsistently seen on [111In]N scans. In contrast, 67Ga accumulation was not affected by abscess age and all abscesses could be identified on a scan 48 hr postinjection. Scans with [111In]N were clearly superior to 67Ga scans for demonstrating early abscesses. Since 67Ga accumulated to a greater extent than [111In]N in abscesses 7 days old, it may be a superior imaging agent for older abscesses.     

L-3-123I-alpha-methyltyrosine for melanoma detection: a comparative evaluation

L-3-123I-alpha-methyltyrosine (IMT) was compared to [67Ga]citrate, [203Pb]Tris and [131I]iodochloroquine (ICQ) with respect to their potential as melanoma seeking radiopharmaceuticals in two tumor lines, a malignant melanotic melanoma of the Sofia type and a malignant amelanotic melanoma of the Greene-Harvey type, transplanted onto Syrian Golden hamsters. [203Pb]Tris and ICQ showed significant accumulation only in melanotic melanoma. In contrast, [67Ga]citrate and IMT accumulated in both tumor lines. [67Ga]citrate has very high tumor-to-skin, tumor-to-eye, and tumor-to-blood ratios at 48 h after injection, but has low selectivity for melanoma and unfavorable physical (high gamma-energy) and biological (half-time) characteristics. IMT has a convenient gamma-energy at 159 keV and a short biological half-time. Maximum of melanoma accumulation is reached at 1-2h after application; tumor-to-tissue ratios are suitable for scintigraphy.     

Colon carcinoma immunoscintigraphy by monoclonal anti-CEA antibody labeled with gallium-67-aminooxyacetyldeferroxamine.

Previous experimental results in nude mice showing that radiolabeling the monoclonal antibody anti-CEA 35 with 67Ga-aminooxyacetyldeferroxamine could give better tumor localization than radioiodination prompted us to initiate the present clinical study. The 67Ga-labeled antibody anti-CEA 35 (185 MBq, 0.7-1.7 mg) was injected preoperatively into 14 patients for colorectal carcinoma imaging. The same antibody labeled with 125I (3.7 MBq, 0.25 mg) was injected simultaneously to compare the 67Ga and 125I dose recoveries in surgical specimens. Twelve of 14 primary tumors gave a positive 67Ga scintigraph. The mean %ID/g recovered in all tumors 3-9 days after injection was significantly higher for 67Ga (0.019%) than for 125I (0.005%) (p < 0.001, paired t test). The tumor-to-normal tissue ratios were generally higher for 67Ga, with the exception of liver. We conclude that 67Ga-aminooxyacetyldeferroxamine improved immunoscintigraphy outside the liver, particularly in the pelvic region. We also show that deferroxamine infusion accelerates the excretion of 67Ga in eight patients and propose that this could lead to further improvement of immunoscintigraphy.     

Uptake and excretion of 67Ga-citrate in malignant tumors and normal cells

Using in vivo and in vitro experimental models, the uptake and excretion of ⁶⁷Ga-citrate in tumor cells and normal cells were studied. The timelapse accumulation of ⁶⁷Ga in the tumor of rats bearing Yoshida sarcoma reached its peak 24 h after the administration of ⁶⁷Ga and gradually decreased thereafter. However, the excretion of ⁶⁷Ga from the tumor was less than that from normal lung. For culture cells in vitro, the uptake of ⁶⁷Ga increased with lapse of contact time between ⁶⁷Ga and the cells, but there was no distinct difference between the results for tumor cells and normal skin fibroblasts. The excretion of ⁶⁷Ga from the cells tended to decrease with prolongation of the contact time, the excretion from tumor cells being only about 10% after a contact time of 24 h. This indicated a significant delay in excretion in comparison with that of normal skin fibroblasts. This delay in the excretion of ⁶⁷Ga may be an important factor in the tumor accumulation of ⁶⁷Ga.     

67Ga accumulation in inflammatory lesion and its mechanism: Comparison with malignant tumor

The accumulation of ⁶⁷Ga in inflammatory lesions increased with time after injection of turpentine iol and reached a plateau 5 days later. At that time the uptake in the lesions was larger than any other tissue, after ten days the lesion uptake decreased. In experiments using rats which had been kept for 5 days after subcutaneous injection of turpentine oil, the accumulation of ⁶⁷Ga in inflammatory lesions increased with time until six days after administration of ⁶⁷Ga-citrate. It is clear from this study that ⁶⁷Ga is avidly accumulated in areas where the subcutanous tissue is infiltrated with neutrophils and macrophages, that it is not accumulated at the sites in which neutrophils are crowded, that nuclear material, mitochondria, lysosomes and microsomes do not play a major role in ⁶⁷Ga accumulation in the lesion and that the main binding acid mucopolysaccharide in the lesion is acid mucopolysaccharide which is none of the following: keratan sulfate, heparan sulfate, heparin, or chondroitin sulfate A, B or C. It is presumed that the main ⁶⁷Ga binding acid mucopolysaccharide is keratan polysulfate (or other oversulfated acid mucopolysaccharides).     

Deferoxamine mesylate enhancement of 67Ga tumor-to-blood ratios and tumor imaging

To improve the tumor-to-blood ratio in ⁶⁷Ga tumor imaging, the effect of administration of deferoxamine mesylate (DFO) was evaluated. DFO improved ⁶⁷Ga tumor-to-blood ratios in tumor-bearing rats. Administration of DFO 12 h after ⁶⁷Ga injection did not decrease the concentration of radioactivity in the tumor of rats, but administration of DFO 4 h after ⁶⁷Ga decreased the concentration of radioactivity in the tumor. Serum unsaturated iron binding capacity in rats was transiently increased by DFO administration, but when DFO was administered before ⁶⁷Ga injection the tumor uptake showed rather decreased levels. In human studies, DFO accelerated the excretion of ⁶⁷Ga from the blood, but tumor images were not necessarily improved.