Intralipid Reduces Toxic Side Effects of DACHPt/HANP

Our previous study27 was designed as a “proof-of-concept” study to show that Intralipid 20% pre-treatment can change the RES uptake, bioavailability, and toxicological profiles of DACHPt/HANP. These results are summarized in Table 1, which shows that its uptake in the liver, spleen, and kidney diminishes and that its bioavailability increases when Intralipid is administered 1 hr before intravenous injection of the nanodrug. Notably, with the Pt-nanodrug and Intralipid being metabolized in the liver, the Pt concentrations reach similar level at 72 hr, 10.1 ± 1.6 and 11.8 ± 3.7 μg/g wet weight (p > 0.1), without- and with-Intralipid pre-treatment, respectively. To increase and prolong the effectiveness of Intralipid, the administered dosages, time courses, and frequencies of Intralipid treatment need to be optimized for each chemotherapeutic regimen. Thus, we have designed a multi-dose protocol described in Fig. 1A, with two doses of DACHPt/HANP and six doses of Intralipid.

Figure 1 Treatment protocols for animal experiments. (A) Treatment protocol for using multiple-doses of Intralipid 20% to investigate the delivery of two doses of DACHPt/HANP. Rats (N = 6 for each group, i.e., Intralipid-treated group and control group) were treated with DACHPt/HANP (2 mg Pt/kg) intravenously twice a week (on Days 1 and 5). Also on Days 1 and 5, Intralipid was intravenously administered at a dosage of 2 g/kg 1 hr before the administration of the nanodrug. On Days 2, 3, 6, and 7, the rats were administered 2 g/kg of Intralipid 20% each day. (B) Treatment protocol for using Intralipid to investigate the delivery of three FDA-approved nanodrugs, Abraxane, Marqibo, and Onivyde. Intralipid was administered intravenously at 2 g/kg 1 hr before the treatment with the nanodrug. After 1 hr, Abraxane, Marqibo, or Onivyde was administered intravenously to a rat at the clinical dose (44 mg/kg, 0.38 mg/kg, and 11.86 mg/kg, respectively). The second dose of Intralipid (2 g/kg) was administered 24-hr post nanodrug treatment. For Abraxane experiment, N = 4 for each group, i.e., the Intralipid-treated group and the control group; for Marqibo and Onivyde experiment, N = 6 for each group. In all the animal experiments, PBS was administered to the control animals. Full size image

Changes of the body weight caused by the administration of DACHPt/HANP and upon treatment with Intralipid 20% are shown in Fig. 2A (N = 6 for each group, i.e., Intralipid-treated and PBS-treated control groups). The first dose of DACHPt/HANP caused animal body weight to decrease ~4% in the 24-hr post administration, followed by slow recovery. The second dose of the Pt-containing nanodrug has caused body weight to decrease for 48 hr before slow recovery. There is no statistically significant difference between the Intralipid-treated group and the control group (no Intralipid treatment). The growth of the body weight of the naïve rats is shown for comparison (dash line in Fig. 2A).

Figure 2 Intralipid treatment reduces the toxic side effects of DACHPt/HANP on day 9. (A) Changes of the body weight of rats upon administration of DACHPt/HANP, with and without treatment of Intralipid (N = 6). (B–M) Light microscopic images of H&E-stained (B,C,F,G, and J,K) and TUNEL-stained (D,E,H–I and L,M) spleen tissue sections. (B,E) are observed from the spleen-tissue sections of DACHPt/HANP administrated, but no Intralipid-treated, animals. (F–I) are from the Intralipid-treated animals. (J–M) are from the spleen-tissue sections of the naïve SD rats. (C,E,G,I,K and M) are the enlarged views of (B,D,F,H,J and L), respectively. Red arrows on (D,E,I and M) indicate the apoptotic cells. An enlarged view of the apoptotic spleen cells is shown as an example in (E). (N–Y) Light microscopic images of H&E-stained and TUNEL-stained kidney tissue sections. Red arrows on (P,Q,T,U and Y) indicate the apoptotic cells. An enlarged view of the apoptotic kidney cells is shown as an example in (Q). (N–Q) are observed from the kidney-tissue sections of DACHPt/HANP administrated, but no Intralipid-treated, animals. (R–U) are from the Intralipid-treated animals. (V–Y) are from the kidney-tissue sections of the naïve SD rats. (Z) Change of the Pt concentration in the kidney as measured by ICP-MS, upon Intralipid treatment, in the multiple-dose experiment. Full size image

Pathological studies [hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay] reveal that the second dose of DACHPt/HANP causes more damages to the spleen, kidney, and liver tissues (Figs 2B–E,N–Q, and S1B–E), comparing with a single dose of DACHPt/HANP as reported in our previous study27. Intralipid treatment significantly reduces the toxic side effects of multi-dose of DACHPt/HANP in the spleen (Fig. 2F–I). With DACHPt/HANP treatment, but no Intralipid, the spleen tissues are characterized by a disorder proliferation of mononuclear cells (Fig. 2B,C) and a large number of apoptotic cells (Fig. 2D,E). An enlarged view of apoptotic spleen cells is shown in Fig. 2E. Upon Intralipid treatment, the spleen cells show better morphology and distribution (Fig. 2F,G vs. B,C). Intralipid treatments significantly reduces the spleen cell apoptosis, especially at the germinal center of the white pulps (Fig. 2H,I vs. D,E). Very few apoptotic cells (red arrows in Fig. 2H,I) are observed in the Intralipid-treated group, comparable to the spleen tissues of the naïve rats (Fig. 2L,M).

The spleen weight of the animals is an indication of the toxic side effects of the nanodrug. As we reported previously27, a single dose of DACHPt/HANP can cause spleen enlargement and swelling. The ratio of the spleen weight/body weight for a naïve Sprague Dawley (SD) rat is 0.31 ± 0.06 (N = 3). Intralipid treatment alone does not change this ratio [0.28 ± 0.02 (N = 3)]. The ratio from a single-dose DACHPt/HANP-treated SD rat is 0.53 ± 0.0827. In our multi-dose experiments, we have found that two doses of DACHPt/HANP, without Intralipid, causes the spleen to shrink and reduces the ratio of spleen weight/body weight to 0.20 ± 0.03 (N = 6). With multi-doses of Intralipid treatment, this ratio is 0.26 ± 0.07 (N = 6). In Fig. S1A, the ratios are shown as the percentage of the normal level.

Nephrotoxicity is a major side effect of platinum drugs32 and our experimental nanodrug, DACHPt/HANP, is developed to reduce the nephrotoxicity27. Although multi-doses of DACHPt/HANP do not cause significant pathological changes in the kidney-tissue sections as shown by H&E staining (Fig. 2N,O), a few apoptotic cells are observed by the TUNEL staining (Fig. 2P,Q). An enlarged view of the apoptotic kidney cells is shown in Fig. 2Q. Intralipid treatment reduces the kidney damage caused by this Pt-containing nanodrug (Fig. 2R–U). The number of the apoptotic cells is significantly reduced at both glomerulus and tubules (Fig. 2T,U vs. P,Q). The kidney-tissue sections from the naïve SD rats (Fig. 2V–Y) are shown for comparison. We have found a 27% decrease of the Pt concentration in the kidney upon Intralipid treatment, as measured by inductively coupled plasma-mass spectrometry (ICP-MS) (Fig. 2Z). With DACHPt/HANP administration, but no Intralipid treatment, the Pt concentration in the kidney is 23.3 ± 2.6 (μg/g wet weight) on Day 9 when the animals were sacrificed. With Intralipid treatment, the Pt concentration decreases to 17.1 ± 0.1 (μg/g wet weight). We have also monitored the kidney function by the serum creatinine assay and observed the changes of the creatinine levels upon Intralipid treatment (Fig. S1N). However, from Day 5 to Day 9, we cannot detect significant changes in the serum creatinine levels upon Intralipid treatment.

The pathological and TUNEL analyses of the hepatic tissue sections are shown in Fig. S1B–M. A large amount of necrosis cells (black arrows on Fig. S1C, which is an enlarged view of Fig. S1B) and the apoptotic cells (red arrows on Fig. S1E, which is an enlarged view of Fig. S1D) are observed from the liver-tissue sections of the DACHPt/HANP-treated animals. The liver tissue sections from the naïve SD rats are shown for comparison (Fig. S1J–M). Probably because the liver damage caused by the second dosage of DACHPt/HANP was too severe, under the current experimental conditions, we have not observed significant improvements in the liver tissue upon Intralipid treatment. We have also monitored the changes in the liver function by the serum alanine aminotransferase (ALT) activity assay (Fig. S1O). From Day 5 to Day 9, we cannot detect significant changes in ALT levels upon the Intralipid treatment.

Intralipid Reduces Toxic Side Effects of Abraxane

We have applied our “proof-of-concept” experimental design, as described in our previous study27, with some minor modifications, to test the effects of Intralipid 20% on the side effects of Abraxane. The treatment protocol is described in the Materials and Methods and shown in Fig. 1B. Intralipid 20% was administered to SD rats at the clinical dose (2 g/kg) using the clinical route (i.e., intravenously) 1 hr before i.v. injection of Abraxane (44 mg/kg, clinical dose for breast cancer treatment) and 24-hr post injection of the nanodrug. The tissue samples collected at 72-hr post injection were used for the histological analysis [N = 4 for Intralipid-treated and PBS-control group].

Abraxane affects the animal body weight significantly, which indicates the toxicities of the drug. Animal body weight keeps decreasing by ~11% during the 72-hr post administration of Abraxane (Fig. 3A). At 72 hr, the body weight of the Abraxane-treated animals is ~18% lower than that of the naïve animals (dash line in Fig. 3A). Intralipid treatment could not change the loss of body weight significantly under current experimental conditions.

Figure 3 Intralipid treatment reduces the toxic side effects of Abraxane. (A) Changes of the body weight of rats upon administration of Abraxane, with and without treatment of Intralipid (N = 4). (B–I) Light microscopic images of the spleen tissue sections treated with Abraxane, with or without Intralipid. (J–M) are from the spleen-tissue sections of the naïve SD rats. Spleen tissue sections were stained with H&E (B,C,F,G and J,K) and TUNEL (D,E,H,I, and L,M). In (D,E,H,I and M), red arrows indicate the apoptotic cells. (B–E) are observed from the spleen-tissue sections of the Abraxane administrated, but no Intralipid-treated, animals. (F–I) are from the Intralipid-treated animals. (N–Y) Light microscopic images of the H&E-stained (N,O,R,S, and V,W) and the TUNEL-stained (P,Q,T,U, and X,Y) kidney tissue sections. The red arrows on (Q,U and Y) indicate the apoptotic cells. (Q) Shows an enlarged view of the apoptotic kidney cells. Full size image

Fixed spleen tissue sections are analyzed with the H&E staining (Fig. 3B,C and F,G) and the TUNEL assay (Fig. 3D,E and H,I). With Abraxane administration, but no Intralipid, uneven distribution of mononuclear cells is observed from the H&E-stained spleen tissue (Fig. 3B,C). A large amount of apoptotic cells is observed at both white pulp and red pulp of the spleen tissue from the TUNEL assay (red arrows in Fig. 3D,E). Upon the Intralipid treatment, these damages are significantly reduced. The spleen cells are more evenly distributed in the Intralipid-treated group (Fig. 3F,G). Intralipid significantly reduces spleen cell apoptosis, especially at the white pulps (Fig. 3H,I). The spleen tissue sections from the naïve SD rats (Fig. 3J–M) are shown for comparison.

The decrease of the spleen weight caused by the administration of Abraxane, with or without treatment of Intralipid, are shown in Fig. S2A. The ratio of the spleen weight/body weight from Abraxane-treated SD rat is 0.19 ± 0.01 (N = 4). Intralipid treatment exhibits no significant change on this ratio (0.22 ± 0.03, N = 4). In Fig. S2A, the ratios are shown as the percentage of the normal level.

Intralipid treatment also reduces the toxic side effects of Abraxane in the kidney (Fig. 3N–Y). Although there is no significant pathological change from the H&E-stained kidney tissue (Fig. 3N,O), Abraxane treatment causes the kidney cell apoptosis, especially at the renal tubules, as indicated by red arrows on Fig. 3Q, which is an enlarged view of Fig. 3P. Intralipid administration significantly reduces the amount of apoptotic cells at the renal tubules (Fig. 3T,U vs. P,Q). Very few apoptotic cells are observed from the Intralipid-treated group (Fig. 3T,U), comparable to the kidney tissues of naïve rats (Fig. 3X,Y).

With respect to the liver, Abraxane treatment causes very mild hepatic cell necrosis (Fig. S2B,C) and apoptosis (Fig. S2D,E). There is no significant change upon the Intralipid treatment (Fig. S2F–I).

Intralipid Reduces Toxic Side Effects of Marqibo

Animal body weight reduces significantly when treated with Marqibo (Fig. 4A, N = 6), indicating the toxic side effects of this nanodrug. We have observed a large number of mitotic cells in the liver (black arrows in Fig. S3B,C), and a few in the spleen and the kidney (black arrows in Fig. 4B,C and J,K). In Figs 4C,K and S3C, an enlarged view of the mitotic cell is shown as an example. Intralipid treatment significantly reduces the amount of mitotic cells in the spleen and the kidney for Marqibo administration (Fig. 4F,G and N,O). With Intralipid treatment, the mononuclear cells in the spleen show more uniformly distribution (Fig. 4F,G vs B,C). The size of the spleen is not affected by Marqibo significantly (Fig. S3A). Intralipid appears to reduce the number of mitotic cells in the liver as well, although there is still quite an amount of the mitotic cells in the liver tissue (Fig. S3F,G). The spleen, kidney, and liver tissue sections from the naïve rats are shown in (Figs 3J–M,V–Y and S1J–M) for comparison.

Figure 4 Intralipid treatment reduces the toxic side effects of Marqibo. (A) Changes of the body weight of the rats upon administration of Marqibo, with and without treatment of Intralipid (N = 6). (B–I) Light microscopic images of the spleen tissue sections treated with Marqibo, with or without Intralipid. The spleen tissue sections were stained with H&E (B,C and F,G) and TUNEL (D,E and H,I). In (C), the black arrows point to the mitotic cells and an enlarged view of the spleen mitotic cell is shown. In (D) and (E) The red arrows indicate the apoptotic cells. (E) Shows an enlarged view of the apoptotic cells. (J–Q) Light microscopic images of the (H,E)-stained (J,K and N,O) and the TUNEL-stained (L,M and P,Q) kidney tissue sections. In (K), the black arrows point to the mitotic cells and an enlarged view of kidney mitotic cell is shown. The red arrows on (L,M and Q) indicate the apopto tic cells. (Q) Shows an enlarged view of the apoptotic kidey cells. Full size image

The large amount of the mitotic cells might be explained by the mechanism of Vincristine, which binds tubulin and causes the microtubule depolymerization, metaphase arrest, and apoptotic death of cells undergoing mitosis33. Although Abraxane is also a mitotic inhibitor, we do not observe this large amount mitoses in the tissues when treated with Abraxane (Figs 3B,C,N,O and S2B,C).

We have observed a large amount to apoptotic cells in the spleen and the kidney when treated with Marqibo (red arrows in Fig. 4D,E and L,M). Intralipid greatly reduces the amount of the apoptotic cells in the spleen and kidney (Fig. 4H,I and P,Q). Consistent with the reduction of the mitotic and the apoptotic cells in the kidney, serum creatinine assay also shows the protective effects of Intralipid (Fig. S3J). Very few apoptotic cells are observed in the liver tissue sections (Fig. S3D,E) and we have not detected the change of serum ALT activity when treated with Marqibo (Fig. S3K).

Intralipid Reduces Toxic Side Effects of Onivyde

Onivyde is a prodrug23. After we have observed the beneficial effects of Intralipid for the above three anti-cancer nanodrugs, we decide to test whether Intralipid can reduce the toxic side effects of Onivyde.

Animals show a mild loss of the body weight post treatment of Onivyde (Fig. 5A, N = 6). Intralipid treatment has no significant effects on the body weight. Consistent with a mild loss of the body weight, Onivyde shows mild toxicities in the spleen, kidney, and liver (Figs 5B–E and J–M, and S4B,E). The H&E-stained spleen-tissue sections from Onivyde administrated, without and with Intralipid treatment (Fig. 5B,C and F,G), look similar to the spleen tissue of the naïve rats (Fig. 3J,K). Onivyde treatment causes mild spleen cell apoptosis as shown by the red arrows in Fig. 5E, which is an enlarged view of Fig. 5D. Intralipid treatment reduces the amount of the apoptotic cells (Fig. 5H,I). Onivyde treatment also slightly reduces the size of the spleen (Fig. S4A). The ratio of the spleen weight/body weight from Onivyde-treated SD rat is 0.21 ± 0.02 (N = 6). Intralipid treatment exhibits no change in this ratio (0.21 ± 0.01, N = 6).

Figure 5 Intralipid treatment reduces the toxic side effects of Onivyde. (A) Changes of the body weight of rats upon administration of Onivyde, with and without treatment of Intralipid (N = 6). (B–I) Light microscopic images of the spleen tissue sections treated with Onivyde, with or without Intralipid. The spleen tissue sections were stained with H&E (B,C and F,G) and TUNEL (D,E and H,I). In (E) and (I), the red arrows indicate the spleen apoptotic cells. (J–Q) The light microscopic images of the H&E-stained (J,K and N,O) and the TUNEL-stained (L,M and P,Q) kidney tissue sections. The red arrows on (M and Q) indicate the kidney apoptotic cells. Full size image

There is no significant change in the H&E-stained kidney-tissue sections from the rats treated with Onivyde, without or with Intralipid treatment, compared with those of the naïve rats (Figs 5J,K,N,O vs. 2V,W). A few apoptotic cells are observed at the renal tubules when treated with Onivyde (Fig. 5L,M). Intralipid treatment significantly reduces the number of the apoptotic cells at the renal tubules (Fig. 5P,Q). Serum creatinine level assay cannot detect this change (Fig. S4N). With regard to the liver-tissue sections, very few necrosis cells (Fig. S4B,C) and apoptotic cells (Fig. S4D,E) are observed from the Onivyde-treated group. Intralipid treatment has no harmful effect to the liver tissue (Fig. S4F–I). We have verified this result by testing the liver function using blood serum ALT activity assay (Fig. S4O). There is no significant change in the serum ALT activity from the animals treated with Onivyde with or without Intralipid.

Effects of Intralipid Treatment on the Anti-tumor Efficacy of DACHPt/HANP in a HT-29 Xenograft Mouse Model

We have conducted a study to show the effect of Intralipid on the anti-cancer efficacy and survival rate using an experimental anti-cancer nanodrug, DACHPt/HANP, with a HT-29 human colon cancer xenograft mouse model. When the tumor volume reached sizes of 100 to 200 mm3, mice were randomly divided into four treatment groups (Fig. 6A): (i) vehicle; (ii) Intralipid alone; and (iii-iv) DACHPt/HANP (2 mg Pt/kg), without and with Intralipid 1-hr pre-treatment. Intralipid treatment shows no negative effect on the animal body weight (Fig. S5). As shown in Fig. 6B, the animal survival rate is 100% for groups (i–iv). Thus, Intralipid treatment exhibits no harmful effect for the survival rate.

Figure 6 Effects of Intralipid treatment on the tumor growth and the anti-tumor efficacy of DACHPt/HANP as well as the survival rate in a HT-29 xenograft tumor model. (A) Treatment protocol. (B) Survival rate of HT-29 bearing BALB/c nude mice for the four treatment groups. (C) Changes of the tumor size upon treatment with vehicle, Intralipid alone, and DACHPt/HANP at 2 mg Pt/kg with or without Intralipid administration. H&E staining of the bone marrow is shown in (D–K). (D,E) DACHPt/HANP treatment, with no Intralipid, causes moderate/severe depletion of erythroid and myeloid precursor cells in the bone marrow. (F,G) Intralipid treatment significantly reduces this damage. (H,I) and (J,K) Bone marrow from (i) vehicle-treated, and (ii) Intralipid alone group. Full size image

The changes of the tumor volume for the groups: (i) vehicle; (ii) Intralipid alone; (iii–iv) DACHPt/HANP (2 mg Pt/kg), without and with Intralipid 1-hr pre-treatment, are shown in Fig. 6C. First of all, there is no significant difference in the tumor volume when we compare the vehicle-treated and the Intralipid-alone group. Intralipid does not promote tumor growth and is a safe agent for the mice tested to reduce toxic side effects of the drug. Second, when we compare the treatment groups (iii) DACHPt/HANP and (iv) the Intralipid pre-treatment before injecting DACHPt/HANP, there is no significant difference in the tumor volume. Thus, Intralipid treatment does not reduce the anti-tumor efficacy of the nanodrug.

In this experiment, we have also found that Intralipid treatment can reduce the toxicities in the bone marrow, which is also an important organ of the RES, caused by the Pt-nanodrug (Fig. 6D–G). The degree of lesions in bone marrow is graded from one to five depending on severity34: Grade 1 = minimal (<1%); Grade 2 = slight (1–25%); Grade 3 = moderate (26–50%); Grade 4 = moderate/severe (51–75%); Grade 5 = severe (76–100%). DACHPt/HANP causes severe damage, i.e., Grade 4, to the bone marrow of the mice, exhibiting moderate/severe depletion of erythroid and myeloid precursor cells (Fig. 6D,E). Upon Intralipid treatment, these damages are greatly reduced and the severity is graded as Grade 2~3, i.e., slight to moderate, (Fig. 6F,G). H&E-stained bone marrow from the vehicle-treated and the Intralipid alone group are shown for comparison (Fig. 6H–K).