A New Approach to Reduce Toxicities and to Improve Bioavailabilities of Platinum-Containing Anti-Cancer Nanodrugs

Platinum (Pt) drugs are the most potent and commonly used anti-cancer chemotherapeutics. Nanoformulation of Pt drugs has the potential to improve the delivery to tumors and reduce toxic side effects. A major challenge for translating nanodrugs to clinical settings is their rapid clearance by the reticuloendothelial system (RES), hence increasing toxicities on off-target organs and reducing efficacy. We are reporting that an FDA approved parenteral nutrition source, Intralipid 20%, can help this problem. A dichloro (1, 2-diaminocyclohexane) platinum (II)-loaded and hyaluronic acid polymer-coated nanoparticle (DACHPt/HANP) is used in this study. A single dose of Intralipid (2 g/kg, clinical dosage) is administrated [intravenously (i. v.), clinical route] one hour before i.v. injection of DACHPt/HANP. This treatment can significantly reduce the toxicities of DACHPt/HANP in liver, spleen, and, interestingly, kidney. Intralipid can decrease Pt accumulation in the liver, spleen, and kidney by 20.4%, 42.5%, and 31.2% at 24-hr post nanodrug administration, respectively. The bioavailability of DACHPt/HANP increases by 18.7% and 9.4% during the first 5 and 24 hr, respectively.

A major limitation for both approved and in-development nanodrugs is their rapid clearance by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system, in particular liver and spleen, which can increase their toxicity to these off-target organs and reduce their efficacy 13,15,22 . Strategies that decrease RES uptake and increase the bioavailability of nanomedicines can improve tumor targeting and decrease the side effects. Many studies have been conducted to decrease RES clearance and to increase the tumor targeting of nanomedicines by modifying nanoparticle characteristics, such as the size, shape, charge, surface property, and composition [23][24][25][26][27][28] . Unfortunately, the total accumulation of the anti-cancer nanodrugs in the tumor represents a small fraction of total injected dose (1-10%). The majority (40-80%) of the injected nanomedicines end up in the liver and spleen 22 . Moreover, each new modification calls for thorough toxicity, pharmacology, and biomechanics studies before translating to a clinical setting.
Our strategy is to target the RES to temporarily blunt the uptake, i.e., to decrease the toxicities in liver and spleen and to increase the bioavailability of nanodrugs using Intralipid 20%, an FDA-approved fat emulsion used as parenteral nutrition source. The rational for this hypothesis is that the infusion of Intralipid has been reported to inhibit RES function by possibly inhibiting peritoneal clearance and impairing the phagocytic activity of Kupffer cells 29 . Kupffer cells in the liver play an important role in the uptake and metabolism of Intralipid 30 . Our recent findings also support this hypothesis. We have found that, in rodents, Intralipid can reduce RES uptake ~50% and increase blood half-life (t 1/2 ) ~3-fold of nano-and micron-sized superparamagnetic iron-oxide particles 31,32 .
We have carried out this study with an improved Pt anti-cancer nanodrug, DACHPt-incorporated nanoparticles (NP), coated with hyaluronic acid polymer (HA) (DACHPt/HANP). We have found that a single, clinical dose of Intralipid (2 g/kg) can significantly reduce the toxic side effects of our Pt-containing nanodrug in liver, spleen, and kidney. Notably, our findings indicate that Intralipid pre-treatment decreases spleen enlargement, which has been reported as a serious side effect of Abraxane ® . A single dose of Intralipid can decrease Pt accumulation in the liver (by 20.4%), spleen (42.5%), and kidney (39.3%) at 24-hr post nanodrug administration. Consequently, the bioavailability of the Pt-nanodrug increases by 18.7% during the first 5 hr and by 9.4% during 24 hr, respectively.
Intralipid Reduces Toxic Side Effects of Pt-Containing Nanodrug. Intralipid 20% was administered to Sprague Dawley (SD) rats at the clinical dose (2 g/kg) using the clinical route (i.e., intravenously) one hour before i.v. injection of DACHPt/HANP. At 24-and 72-hr post injection of DAHPt/HANP, blood samples were collected to determine serum alanine aminotransferase (ALT) activity and creatinine level to investigate liver and kidney damages. The tissue samples collected at 72-hr post injection were used for histological analysis. The tissue samples from naïve (SD) rats were used as controls.
Pathological Analysis and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay for Apoptotic Cells in Liver. Light microscopic images of hematoxylin/eosin (H & E) stained liver tissue sections are shown in Fig. 1A-C,F-H,K-M. Images of TUNEL stained liver tissue sections are shown in Fig. 1D,E,I,J,N,O. With DACHPt/HANP administration, but no Intralipid pre-treatment, the pathological changes in the liver tissue are characterized by necrosis, as indicated by black arrows in Fig. 1C, which is an example of enlarged view from Fig. 1A,B. Apoptotic cells are observed with TUNEL staining, as indicated by red arrows in Fig. 1D,E, from the liver tissue of this treatment group. An enlarged view of an apoptotic cell is shown as an example in Fig. 1E. These damages are significantly reduced upon Intralipid pre-treatment. The liver tissue sections from the Intralipid pre-treated group are shown in Fig. 1F-J. Very few cell necroses (black arrow in Fig. 1H) and apoptotic cells (red arrows in Fig. 1J) are observed, comparable to the liver tissues of naïve rats (Fig. 1K-O). Spleen Enlargement. Spleen swelling and enlargement are observed from DACHPt/HANP-treated animals, when the animals are sacrificed 72-hr post nanodrug administration ( Fig. 2A left). Intralipid pre-treatment appears to reduce spleen swelling ( Fig. 2A right). The ratio of spleen weight/body weight for a naïve Sprague Dawley (SD) rat is 0.31 ± 0.06 (n = 3). Intralipid treatment does not cause spleen swelling with the ratio of 0.28 ± 0.02 (n = 3). The ratio from a DACHPt/HANP treated SD rat is 0.53 ± 0.08 (n = 3). Upon Intralipid pre-treatment, this ratio reduces to 0.4 ± 0.008 (n = 3). In Fig. 2B, the ratios are shown as the percentage of the normal level.

Changes of DACHPt/HANP Accumulation in Tissues upon Intralipid Pre-Treatment. The Pt
concentration in tissue (spleen, liver, and kidney) and blood of naïve animals or Intralipid along or phosphate-buffered-saline (PBS) treated animals is below 0.01 part per million (ppm).
With the drug being metabolized in the liver, the Pt concentrations reach similar level at 72 hr: 10.1 ± 1.6 and 11.8 ± 3.7 (μ g/g wet weight), without-and with-Intralipid pre-treatment, respectively.

Discussion
After several decades, the research seeking for less toxic Pt drugs and better drug delivery methods, which can decrease the associated side effects and improve the anti-cancer efficacy as well as the quality of life of the patients, still goes on. We have found a new approach to reduce the side effects and increase the bioavailabilities of an anti-cancer Pt-containing nanodrug (DACHPt/HANP), by using an "old" FDA approved agent, Intralipid. Since the approval of cisplatin in 1979, Pt-based drugs, including carboplatin and oxaliplatin (second and third generation), have become the most potent as well as the most widely prescribed anti-cancer drugs 2 . Unfortunately, its continuous use is greatly limited by dose limiting toxicities, partial anti-tumor response in most patients, development of drug resistance, and tumor rela pse 2,3,5,8-10,33 . Nanocarrier-based drug delivery may generate new therapeutic approaches for Pt-drugs. Pt-based nanodrugs are providing encouraging preclinical and clinical results and may facilitate the development of the next generation of Pt chemotherapy 18,19,34 . However, the important questions of how to decrease the RES uptake, which accounts for 40-80% of injected nanodrugs, and how to reduce the toxic side effects caused by this off-target uptake, still need answers. Our studies show that Intralipid pre-treatment can be used to reduce RES uptake and side effects, and improve the bioavailability and clinical applications of Pt-containing nanodrugs. Moreover, we have observed that Intralipid treatment can decrease Pt accumulation in kidney, thus reducing nephrotoxicity of the Pt drug.

Figure 5. Effects of Intralipid pre-treatment on the serum ALT activities (A) and creatinine levels (B)
in DACHPt/HANP treated rats. When the rats are treated with 4 mg Pt/kg of the nanodrug, Intralipid pretreatment group shows significantly lower serum ALT activity and creatinine level (A,B). The group, which is pre-treated with Intralipid followed by the treatment of a higher dosage (6 mg Pt/kg) of DACHPt/HANP, exhibits lower ALT activity (A) and creatinine level (B) than the group, which is treated with 4 mg Pt/kg of the nanodrug, but no Intralipid. * p < 0.001; ** p < 0.05. spleen would cause toxic side effects. For many nanomedicines, the toxicity in the mononuclear phagocyte system is the killer for further development 35 . DACHPt loaded in HANP induces cancer cell apoptosis by causing cross-linking of DNA and DNA-protein. DACHPt-loaded polymeric micelles have been reported to cause liver toxicity 20 . When animals were sacrificed at 72-hr post DACHPt/HANP administration, we observed dramatic swelling and enlargement of the spleen from DACHPt/HANP-treated animals. Pre-treatment with Intralipid 20% (clinical dose, 2 g/kg) can reduce spleen swelling significantly (Fig. 2). Pathological and cell apoptosis analyses reveal that Intralipid can be used to decrease the toxic side effects of our anti-cancer nanodrug in the mononuclear phagocyte system (Figs. 1 and 3). The serum ALT assay also indicates that Intralipid can protect liver from the damage caused by the nanodrug off-target accumulation (Fig. 5A).
In a previous study 31 , we have found that in rodents, Intralipid can reduce RES uptake by ~50% of nano-and micron-sized particles in which MRI contrast agents are loaded. The RES plays an important role in the uptake and metabolism of Intralipid 30,36 . The blood half-life of Intralipid 20% administered by intravenous bolus in rats is 8.7 ± 3.0 min 30,36 . The diameter of the Intralipid particles range from 200 to 1000 nm 37 . As shown in Fig. 6A,B, Intralipid pre-treatment decreases the liver and spleen uptake of the nanodrug by 20.4% and 42.5% at 24-hr post nanodrug administration, respectively.
Interestingly, Intralipid pre-treatment can also decrease the Pt accumulation in the kidney (Fig. 6C1,C2). Nephrotoxicity is one of the most severe side effects of current Pt drugs 2,3,6,8-10 . DACHPt/ HANP nanodrug is designed to increase the concentration and prolong the half-life of DACHPt at tumor sites and to decrease the side effects like nephrotoxicity. Although our Intralipid therapy was originally designed to decrease the RES uptake of the nanodrug, Intralipid pre-treatment could also decrease the Pt drug (DACHPt and/or DACHPt/HANP) accumulation in kidney by 28.7% at 72 hr. Regarding the Pt concentration, we should keep in mind that two components contribute to the Pt concentration: the DAHPt/HANP nanodrug and the DAHPt molecule, which is released from the polymer coating. As a consequence, Intralipid also decreases the nephrotoxicity of the Pt-nanodrug (Figs. 4 and 5B).
This protective effect of Intralipid is so potent that the rats from a higher dosage treatment (6 mg Pt/kg of DACHPt/HANP) exhibit a less hepatocellular and nephrocellular damages (Fig. 5A,B). This indicates that, using Intralipid, the clinicians might be able to give the patients more anti-cancer nanodrugs to kill the tumors with less toxic side effects! Intralipid can change the clearance and increase the bioavailability of the nanodrug. Our results show that a single dose of Intralipid can increase the bioavailability of DACHPt/HANP by 18.7% during the first 5 hr (Fig. 7). It has been reported that after i.v. administration of Intralipid, the circulating ketone bodies increased ~100% in 30 min, which indicates an active metabolism of Intralipid by the liver 30 . This active metabolism might explain the decrease of the effectiveness of Intralipid after 5 hr. To increase and prolong the effectiveness of Intralipid, the administration routes, dosages, and time courses of Intralipid treatment need to be optimized in a future study. Multiple administrations of Intralipid may be necessary.
Moreover, the development of targeted nanomedicine has made an important impact in new drug development in neurology 38 , cardiology 39 , and inflammation 33 . The EPR effect is found not only in cancer, but also in a wide range of inflammatory diseases, such as atherosclerosis [40][41][42] . Thus, our findings for Intralipid pre-treatment could have broad applications besides cancer.

Concluding Remarks
Our study shows that Intralipid can be used to reduce the toxic side effects of Pt-containing anti-cancer nanodrugs in the liver, spleen, and kidney, and also to improve the bioavailability of the nanodrug. Our approach is also a general one applicable to any approved and in-development nanodrugs to improve their bioavailability and to decrease their toxic side effects, without any new modification of the nanodrugs and/or the nanocarriers. Intralipid has been used for over 40 years as a safe source of parenteral nutrition for patients and so can readily translate to clinical use. The outcome of this study has the potential to decrease the toxic side effects of anti-cancer nanodrugs and other nanodrugs, and therefore to reduce human suffering. Also, increasing efficacy could lead to a reduction of the dosage of these expensive drugs: the average cost per dose is US$4,000-6,000. Thus, our findings for the use of Intralipid with nanodrugs can also lead to the reduction of healthcare costs as well as to the improvement of the quality of life for patients who undergo the therapeutic treatment. Preparation and Physical Properties of DACHPt/HANP. DACHPt/HANP was prepared with modified procedures from a previously described method 18 . In brief, DACHPtCl 2 was mixed with silver nitrate ([AgNO 3 ]/[DACHPt] = 2) to form an aqueous complex. The solution was kept in the dark at 25 °C for 24 hr. AgCl precipitates were removed by centrifugation followed by filtration through a 0.22-μ m hydrophilic polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). Subsequently, HA/ Boc-His/PEG graft copolymers, comprising hyaluronic acid (Mw = 16 kD), were added to the aqueous complex of DACHPt at a 0.33 molar ratio of DACHPt to carboxylate groups of the HA modified polymers. The mixture was stirred in the dark for three days at 25 °C. The reaction mixture was sonicated and then purified by ultrafiltration against deionized water to remove uncoordinated DACHPt. The product was filtered through a 0.22-μ m PVDF membrane and lyophilized with 10% trehalose.

Materials and
The particle size and PI of DACHPt/HANP was determined by dynamic light scattering using a ZetaPlus (Brookhaven, Holtsville, NY). Zeta potential was measured by the laser Doppler anemometry (Zeta Plus zeta potential analyzer, Brookhaven Instruments Corporation). TEM images were taken by using a Cryo transmission electron microscope (Cryo-TEM) [JEM-2100 (JEOL, Tokyo, Japan)] operated at 200 kV with attachment of energy dispersive spectroscopy (EDS). A droplet of DACHPt/HANP solution was adsorbed on a cleaned carbon film supported copper grid. After excess sample was removed, phosphotungstic acid (Merck) was used as negative stain reagent to improve the image contrast. TEM grid was dried in the contamination-free environment and reserved in the electronic dry cabinet for further TEM analysis.
Encapsulation Efficiency of DACHPt in DACHPt/HANP. In order to determine the encapsulation efficiency of DACHPt in the nanocomplex, the amount of Pt were quantified by inductively coupled plasma-optical emission spectrometry (ICP-OES) in preparation processes. Encapsulation efficiency (EE %) was calculated using below formula: where W P is the total amount of Pt after purification by passing through a 0.22 μ m filter and W T is the total quantity of Pt determined before purification.
Experimental Design. Male SD rats, with body weights between 250 and 280 g, were used. Intralipid 20% was administered by intravenous injection at a clinical dose of 2 g/kg. PBS was administered to control animals. After one hr, DACHPt/HANP (2 mg Pt/kg for bioavailability and biodistribution studies, n = 14 for Intralipid pre-treatment group and n = 14 for control group; 4 mg Pt/kg for toxicity studies, n = 3 for Intralipid pre-treatment group and n = 3 for control group; 6 mg Pt/kg for another toxicity study to test the serum ALT activity and creatinine level, n = 3 for Intralipid pre-treatment group) was injected intravenously. Blood samples were collected at different time points to determine the bioavailability of DACHPt/HANP. At 5-, 24-, and 72-hr post injection of DACHPt/HANP, tissues (liver, spleen, and kidney) were collected for the Pt-level determination. The tissue samples collected at 72-hr post injection were used for histological analysis.

Blood
Bioavailability. An indwelling jugular vein catheter was used for repeated blood samplings.
Blood samples (100 μ L) were collected at different time points to determine the changes of bioavailability of DACHPt/HANP upon Intralipid treatment. Blood was sampled after DACHPt/HANP injection at 1, 5, 10, 20, 45, and 60 min, 3, 5, 24, 28, 48, 52, and 72 hr. The blood samples were decomposed in HNO 3 (0.5 mL) at 60 °C overnight and re-dissolved in 0.5 mL of 2 N HCl 18,20,43 . Suitable dilutions were prepared using 5% HCl to reach a final Pt concentration in the range of 0.02 to 1 part per million (ppm). Samples were analyzed for Pt concentration by inductively coupled plasma-mass spectrometry (ICP-MS) [NexION 300X (PerkinElmer, Waltham, MA)], with modified procedures from our previous studies 31,32 . 194 Pt, 195 Pt, and 196 Pt isotopes were analyzed and similar results were obtained from the measurement of these three isotopes. The Pt concentrations shown in this manuscript were calculated from the measurements of 194 Pt. Bioavailability was calculated by the area under the curve (AUC), namely the integral of the concentration-time curve, using the trapezoidal rule with the use of KaleidaGraph 4.1 (Synergy Software, Reading, PA).
Pt Levels in Tissues. The wet weight of each tissue sample was recorded. Tissue homogenate (0.5 mL) was decomposed in HNO 3 (1 mL) at 60 °C overnight. The rest of the tissue was fixed in 4% paraformaldehyde for histological analyses. The HNO 3 -digested samples were evaporated and then re-dissolved in 0.5 mL of 2 N HCl 43 . The Pt concentrations in the solution were analyzed by ICP-MS as described above.
Pathological Analysis and TUNEL Assay. Histological examinations and TUNEL assays were performed by the Transplantation Pathology Laboratory of the University of Pittsburgh Medical Center (Pittsburgh, PA). Paraffin-embedded 5-μ m sections were stained with hematoxylin/eosin (H & E), or performed TUNEL staining. For histopathological diagnosis, slides were examined by light microscopy and photomicrographs were taken using a Moticam 2300 camera mounted on an Olympus Provis microscope with Mtic Images Plus 2.0 software.
ALT Activity Assay and Creatinine Colorimetric Assay. The activity of ALT in serum was measured by using the ALT Activity Assay Kit purchased from Sigma-Aldrich, according to the supplier's protocol. Serum creatinine level was measured by using the Creatinine Colorimetric/Fluorometric Assay Kit purchased from BioVision. Statistical Analysis. Statistical analysis was carried out with Student's t test. A p value < 0.05 was considered statistically significant.