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The mean plasma concentration-time profiles of Cy5.

Introduction to Pharmacology

In contrast, intravenous injection of micelles carrying Cy5. These PK parameters are summarized in Table 1. We observed distinct differences in the PK profiles of Cy5. This increase in half-life by micellar formulation can be attributed to protection of let-7b mimic from enzymatic degradation as well as a decrease in renal clearance. The C max of micellar Cy5. The CL T of Cy5-let-7b was The V d, Z of Cy5. These results clearly demonstrate the superiority of micelles for miRNA delivery in orthotopic pancreatic mouse models as compared to the emulsion formulation.

The biodistribution of let-7b mimic in major organs including liver, spleen, tumor, kidneys, lungs, and heart was also quantified. While the whole-body and ex vivo imaging provided qualitative data, let-7b concentration in each tissue was further determined by measuring fluorescence of extracted Cy5.

Mice treated with the emulsion showed higher Cy5. In case of the micellar formulation, Cy5. Fluorescence intensity of urine samples and plasma concentration of Cy5.

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At indicated time points 0. B Plasma concentration of Cy5. C Percentage of injected dose of Cy5. Plasma pharmacokinetic parameters of Cy5. PK parameters were estimated by non-compartmental analysis using Phoenix WinNonlin software. Biodistribution of Cy5. Inset graphs are the same data emphasizing the tumor data that appears in the bar graph.

PK parameters for major organs for both emulsion and micelle formulations are summarized in Table 2. In the tumor, there was a 2. Since oligonucleotides are highly susceptible to degradation by serum ribonucleases, the observed fluorescence may come from degraded Cy5. Therefore, we carried out real-time RT-PCR to quantify the full-length let-7b in the tumor samples at each time point in treated and untreated mice. Untreated tumor samples were used to normalize the miRNA concentrations. Percentage of injected dose ID of Cy5.

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Tumor distribution of Cy5. A Relative let-7b levels in tumors at different time points. B Plasma concentration of GDC C Percentage of injected dose of GDC Biodistribution of GDC in different organs after single intravenous injection. We also determined the plasma and organ PK profiles of GDC following intravenous injection of the emulsion and micelle formulations for the co-delivery of GDC and Cy5.

For both treatment groups, GDC was rapidly eliminated from the circulation and distributed to all major organs after systemic administration, with higher accumulation in the liver Figure 6 B and Figure 7. Peak plasma concentration C max of GDC was 2-fold higher for the micelles-treated group than that of the emulsion-treated group: We observed a 2.

Biodistribution of GDC in major organs like liver, spleen, tumor, kidneys, lungs, and heart was also determined, and results are shown in Figure 7. At the initial time point post-injection 0. Overall, heart, lung, and spleen showed the lowest drug deposition among the organs analyzed, which also dropped slowly with time. However, we did not observe a significant difference in concentrations between the emulsion- and micelles-treated groups in these organs.

For the emulsion formulation, GDC accumulation in the kidney was high at two early time points and then showed a sharp decline, whereas in the micelles-treated group, GDC accumulated slowly in the kidney and persisted up to the 4 h time point. As expected, GDC accumulation in the liver was remarkably higher than other organs for both formulations. However, the liver concentration increased overtime in the micelle-treated group, whereas it declined with time in case of the emulsion group.

GDC accumulated continuously at the tumor site with the highest observed concentrations 4 h post-injection, then decreased gradually, but still detectable for up to 24 h post injection. At the initial time points 0. On the other hand, micelles showed 4. At the 4 h time point, GDC accumulation in the liver and kidney was 4. The tumor showed a non-significant difference in GDC accumulation at the initial 0. We did not observe a reduction in mean body weight MBW or treatment-associated morbidity in mice, indicating that micellar formulations of both drugs were well tolerated.

The treatment groups had reduced size and weight of the primary tumors compared to the control group. Among treatment groups, combination of GDC and let-7b significantly decreased tumor growth. Quantification of bioluminescence signal from distinct groups is presented in Figure 9 B and tumor size measured one-week post treatment is shown in Figure 9 C-D. A Kidney, B lung, C liver, D tumor, E heart, and F spleen after intravenous administration of emulsion and micellar formulations of Cy5. A Bioluminescence at different times. C Isolated tumor pictures.

D Tumor weight. Ki staining represents the vital areas of the tumors and is a marker of proliferation. Ki staining showed marked differences between different treatment groups. Tumors treated with the micellar formulation of GDC and let-7b revealed reduced Kipositive cells compared to the non-treated or single drug-treated animal groups Figure 10 A. GLi-1 is the effector transcription factor of Hh signaling that drives cancer progression. We assessed the degree of Gli-1 activation in tumors via IHC. We found that Gli-1 was strongly upregulated in the tumor tissue of the control group compared to the group treated with the micellar formulation of GDC and let-7b Figure 10 B.

Gli-1 staining was the least positive in the GDC and let-7b combination treatment group. We next evaluated levels of KRAS in various tumor tissues. Considering that cationic micelles may cause acute systemic toxicity, hematological parameters of orthotopic pancreatic tumor-bearing NSG mice were determined after a single injection of micelle and emulsion formulations carrying GDC and Cy5.

Further, we observed no obvious changes in serum albumin ALB and alanine aminotransferase ALT , but a slight increase in alkaline phosphatase ALP levels, which is non-significant and indicates no cell membrane injury or tissue damage to the liver for the treated groups compared to the control group. No change in the concentration of creatinine, a biochemical marker of kidney damage, was observed in treated animals.

However, exposure to the micelles resulted in a slight but not significant decrease in the concentration of circulating urea and potassium. Sign of any pancreatic toxicity was evaluated by determining amylase AMY levels: increased AMY levels were found in the emulsion-treated group; however, the difference between the control and treated animals was insignificant.


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IHC staining. Next, we investigated and compared the pathological changes in mouse vital organs such as heart, liver, spleen, lung and kidney among the groups at 48 h post administration of micelles in PK and post-therapeutic studies. No disorders or overt, concomitant signs of toxicity were observed.


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Further, there was no evidence of immune cell infiltration in any of these organs of the mice treated with the micelle formulations of GDC, let-7b or their combination. This study presents biodistribution, therapeutic efficacy and toxicity profiles after systemic administration of co-formulated polymeric micelles of polyanionic hydrophilic miRNA let-7b and small molecule hydrophobic drug GDC into orthotopic pancreatic tumor-bearing NSG mice, and the results were compared with those of mice treated with emulsions carrying these miRNA and drug. However, most of the current nanoparticle platforms can deliver either small molecules or oligonucleotides but are rarely used for their co-delivery.

Nanocarriers capable of carrying both small molecules and oligonucleotide are not only rare, but they are also not thoroughly investigated for their in vivo PK and biodistribution properties. Previously, we developed nanocarriers capable of carrying small molecules and miRNA simultaneously and evaluated their efficacy in a subcutaneous tumor model, and a common bile duct ligation CBDL -induced liver fibrosis model [ 4 , 22 ]. The potential challenges associated with NPs include rapid clearance of the particles by the MPS, vehicle-related cytotoxicity, and elicitation of cytokine response due to activation of toll-like receptors TLRs [ 28 ].

For instance, PEG molecule on the surface of the nanocarriers and particle size below nm can decrease serum protein adsorption and prevent MPS recognition, endowing the nanocarriers with longer circulation time [ 29 ]. The micelles used in this study were nm in size and their surface was decorated with PEG, making them suitable for extended circulation and increasing the chance of high accumulation in the tumor. By whole-body fluorescence imaging, mice injected with the emulsion containing Cy5-let-7b and GDC showed Cy5. In contrast, mice treated with micelles carrying Cy5-let-7b and GDC showed significant fluorescence signal co-localized with tumor tumor location was confirmed by luciferase expression Figure S1 , and signals increased with time post-injection Figure 1 and Figure 2 , indicating tumor accumulation of micelles.

Renal excretion remained the major route of miRNA excretion and free miRNA in the emulsion was cleared rapidly from the system and appeared immediately in the urine, resulting in no accumulation in the major organs Figure 3 A.

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After i. In the micelles-treated mice, a high Cy5. We observed low renal excretion at the initial time points due to reduced uptake of large molecules by the kidneys when using nanocarrier-formulated miRNAs [ 31 ]. However, previous studies have also shown that most cationic polymer-based oligonucleotide delivery systems get dissembled in the kidney as the glomerular basement membrane is rich in polyionic heparin sulfate [ 32 ]. Therefore, we observed strong Cy5. A typical biphasic blood circulation profile was observed for both the micellar and emulsion formulations-treated groups Figure 3 B.

An understanding of the distribution of micelles in vital organs is crucial for understanding normal tissue tolerance, and secondary organ toxicities [ 33 ]. The tissue s levels of Cy5. As kidneys are located deep inside the body, their fluorescence was undetectable in the whole-animal imaging [ 34 ]. We observed the highest Cy5. Similarly, a higher concentration of GDC 0. Liver, spleen, and kidney are highly perfused organs and contain fenestrae or endothelial structures allowing large particles to cross from the bloodstream into the organs' interstitium, justifying drug accumulation in these organs [ 35 ].

In contrast, high uptake of micelles in the tumor is due to its enhanced angiogenesis and undeveloped vasculature, resulting in high permeability that allows entry of nanoparticles up to nm. At 4 h post-injection, fluorescence was higher in the tissues than in the blood, indicating slow clearance from the organs.

Quantitative measurement of the fluorescence signal in isolated tissues further confirms the above imaging results, suggesting the micelles could selectively increase drug accumulation in these organs Figure S3. The preclinical PK profiles of a formulation often serve as an early assessment of its potential success in the clinic. We performed PK studies for Cy5.

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Essential Pharmacokinetics: A Primer for Pharmaceutical Scientists

Further, these results are in agreement with the previous findings, where micelles provided slightly longer sustained release and increased MRT of encapsulated drugs [ 36 , 37 ]. Amphiphilic polymeric micelles can reduce drug uptake by the kidney. However, kidney exposure of miRNA 5.

The delayed appearance of miRNA in the urine of micelles-treated mice indicates that this formulation only delayed their elimination but did not modify the elimination route of miRNA.

However, GDC is not known to be significantly eliminated through the kidney [ 37 ]. Interestingly, Cl and V d values for both miRNA and GDC in micelles were lower than those for free emulsion, while the mean residence time was 2-fold higher. The probable reasoning could be sustained release of drugs into circulation upon micellar formulation, resulting in decreased V d [ 39 ], rather than the particle size difference between these formulations Figure S4.

These conclusions are also supported by our previous report, where micelles showed sustained drug release and high kinetic stability in the presence of serum [ 4 ]. However, the fluorescence measurement showed very weak signal at 24 h post administration. The reason for these observations could be the sensitivity difference between the two techniques. Therefore, significantly higher levels of the parent guide strand of miRNA could be determined in the tumor up to 24 h post injection of the micelle formulation by real-time RT-PCR Figure 6 A.

After observing an increase in drug exposure by micelles, we wanted to determine if micelles can deliver effective concentrations to the tumor and provide therapeutic benefits. GDC and let-7b micelles exhibited the best therapeutic efficacy for pancreatic cancer. We observed significant decreases in the silencing of target genes Gli-1 and KRAS specifically in the combination treatment group.

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This might be attributed to the improved profile of GDC and let7b by micelles, increasing the amount of drug deposited in the tumor Figure 9 and Figure These observations are in line with our previous study, which showed that GDC reduces proliferation and induces apoptosis of tumor cells via a Glidependent manner and chemosensitizes them to anti-KRAS miR-let-7b [ 4 ].

Exogenous gene silencing molecules can result in cellular toxicity due to non-specific off-target effects including activation of the immune system. Therefore, we determined the therapy-related toxicity after i. In both single and multiple-dose studies, general toxicity parameters did not reveal any systemic changes by the micelles Figure S5. Hematological parameters were within the normal range, and there was no statistically significant change in animals exposed to micelle and emulsion formulations when compared to the controls. There was a slight decrease in the concentration of circulating glucose and sodium ions.

Levels of calcium and amylase were slightly increased for emulsion-treated mice. All other parameters in hematology or clinical chemistry showed no statistically significant changes. As shown in Figure S6 and Figure S7 , the formulations were well tolerated and did not show any signs of organ toxicity such as ballooning degeneration of hepatocytes, increased alveolar wall thickness and cellular infiltration in the lung, myocardial fibrillar loss and vacuolation in heart tissues, edema, tubular vacuolization with hemorrhagic areas in the kidney, or increased numbers of granulocytes in the spleen.

Our findings here provided us with an excellent picture of the biodistribution and anti-cancer efficacy of let-7b and GDC when co-formulated in micelles and administered systemically in orthotopic pancreatic tumor-bearing mice.

Essential pharmacokinetics : a primer for pharmaceutical scientists - Semantic Scholar

Our results suggest that our formulation can be used as a drug delivery platform to overcome the limitations and adverse effects of gene and anticancer drug delivery. In conclusion, the micelles controlled the biodistribution of Cy5. We have demonstrated here that the fluorescence labeling method for oligonucleotides is applicable for preclinical PK studies of unformulated and micellar formulated miRNAs.

Our results showed that the micellar co-delivery system could accumulate efficiently in the tumor, and more importantly both the encapsulated drugs showed similar biodistribution patterns when injected as micelles. Micelles loaded with GDC and let-7b showed high tumor regression compared to single drug-loaded micelles. Micellar formulation of GDC and let-7b could improve therapeutic efficacy in vivo by increasing intracellular drug concentrations.

Although our toxicity and immunostimulatory tests are quite encouraging, additional studies including long-term toxicology and safety studies will be needed to take this potential delivery system to the next level of drug development. Y performed the implantation surgeries and collected data. All authors gave their final approval. Desmoplasia of pancreatic ductal adenocarcinoma. Clin Gastroenterol Hepatol. A paracrine requirement for hedgehog signalling in cancer. Inhibition of hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.

Codelivery of small molecule hedgehog inhibitor and miRNA for treating pancreatic cancer. Mol Pharm. Let-7b and miR are down-regulated in tumor tissue and correlate with microvessel density and survival outcomes in non-small-cell lung cancer. PLoS One. Regression of murine lung tumors by the let-7 microRNA. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther.

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