Therapeutic potential of human serum albumin nanoparticles encapsulated actinonin in murine model of lung adenocarcinoma

Abstract

Non-small cell lung cancer comprises 85% of the global lung cancer cases. Conventional chemotherapeutics possess certain limitations like systemic toxicity and drug resistance that requires the development of new therapeutic agents for successful treatment of lung cancer. Actinonin, a human peptide deformylase inhibitor, has demonstrated anti-cancerous properties in various leukemias and solid cancer types. However, it has limited therapeutic application because of its low bioavailability and systemic toxicity if administered in free form. This limitation can be overcome by using nano-delivery systems that will increase the therapeutic efficacy of actinonin. In the present study, human serum albumin actinonin nanoparticles were prepared using a desolvation technique and folic acid was conjugated to lysine residues of albumin for effective delivery to the lung. The lung adenocarcinoma model was established 24 weeks after intraperitoneal administration of urethane and chemotherapeutic efficacy of free as well as nanoencapsulated actinonin was evaluated. This study demonstrated anti-proliferative potential of folic acid conjugated human serum albumin nanoparticles encapsulating actinonin. The intraperitoneally administered nanoformulation exhibited sustain release profile of actinonin with longer half-life and mean retention time. The reduced dose frequency resulted in therapeutic efficacy comparable to free drug in vivo in terms of 100% survival and reduced tumor burden along with downregulation of epidermal growth factor receptor, folate receptor α and peptide deformylase expression in lung adenocarcinoma mice model. Therefore, actinonin encapsulated albumin nanoparticles-based therapy holds great potential as an alternative strategy to improve its anti-cancerous activity against lung adenocarcinoma.

Keywords: Non-small cell lung cancer; actinonin; adenocarcinoma; folate; human serum albumin; peptide deformylase.

Figure 1.

The characterization of developed actinonin nanoparticles. (a) Representative size distribution of actinonin nanoparticles. The three independent formulations are represented in three different colors. (b)Scanning electron microscopic images of actinonin nanoparticles at 70k X magnification. (c) Transmission electron microscopic image of folate conjugated actinonin nanoparticles. The FTIR data of (d) empty HSA nanoparticles, (e) free actinonin, (f) actinonin nanoparticles. The x-ray diffraction analysis of free drugs and nanoformulated drugs. (g) ENP-empty nanoparticles without folate, (h) FA-folic acid-NHS conjugate, (i) F ACT- free actinonin, (j) ANP- actinonin nanoparticles, The y-axis shows the arbitrary intensity units and x-axis – 2θ˚ (angle in degrees). The experiments were performed in triplicate.

Figure 2.

Development of lung adenocarcinoma 24 weeks model. The representative images of lungs (a) control, (b) H&E of tumor lesion at 8th week of latency, (c) CK7^- 8th week of latency tumor lesion, (d) gross tumor lesion at 16th week of latency,(e) H&E of tumor lesion at 16th week of latency, (f) H&E of lung tumor lesion at 24th week of latency, (g) CK7^- 24th week of latency tumor lesion, (h) Napsin A^- 24th week of latency tumor lesion, (i) TTF-1⁺24th week of latency tumor lesion.

Figure 3.

Survival analysis. The survival chart for free and nanoformulated actinonin-treated groups for both latency periods. UT – untreated, AFI – free actinonin i.p and ANI – nanoformulated actinonin i.p. 6 and 10 indicate the latency months.

Figure 4.

Gross lung analysis. (A) Representative gross lung tumor lesions of (a) untreated group; (b) free actinonin-treated group; (c) nanoformulated actinonin-treated group; at 6 months latency period. Representative gross lung tumors of (d) untreated group; (e) free actinonin-treated group; (f) nanoformulated actinonin-treated group at 10 months latency period. (B) The tumor number for (a) 6 months latency period groups and (b) 10 months latency period groups. The tumor volume for (c) 6 months latency period groups and (d) 10 months latency period groups. UT – untreated, AFI – free actinonin i.p and ANI – nanoformulated actinonin i.p. Circles highlight the tumor lesions. Values are mean ± S.E. of 3–5 animals.

Figure 5.

Immunohistochemistry and flow cytometric analysis of lung adenocarcinoma lesions. (A) Representative images of TTF-1⁺ lung tumor lesions at (a) 6 months latency period and (b) 10 months latency period. (B) Representative H&E lung tumor lesions of (a) control group; (b) untreated group; (c) free actinonin-treated group; (d) nanoformulated actinonin-treated group. H&E of other vital organs (e) kidney, (f) liver and (g) spleen. Black arrows indicate lymphocytic infiltration and yellow arrow indicate apoptosis.(C) The excised tumors screened for presence of EpCAM and CD45 positive cells. (a) Control EpCAM⁺, (b) Tumor EpCAM⁺, (c) Control CD45⁺, (d) Tumor CD45⁺.

Figure 6.

Flow cytometric analysis of lung adenocarcinoma lesions. Percentage of population representing necrosis and apoptosis in actinonin-treated animal group of (a) 6 months latency period and (b) 10 months latency period. The mean fluorescence units representing ROS levels in actinonin treatment group of (c) 6 months latency period and (d) 10 months latency period. (e) TEM image of NCI-A549 cellular uptake of actinonin nanoparticles. (f) TEM image of NCI-A549 cells exposed to actinonin nanoparticles for 48 h. Values are mean ± S.E. of 3–5 animals. UT – untreated, AFI – free actinonin i.p and ANI – nanoformulated actinonin i.p. ***p ≤ 0.01; **p ≤ 0.01; *p ≤ 0.05.

Figure 7.

Selection of population based on mitochondrial membrane potential. (a) Scatter plot with all subpopulations, (b) cells with higher to intermediate fluorescence intensity (P1), (c) cells with lowest fluorescence intensity (P2), (d) cells with highest fluorescence intensity (P3), (e) tabulated subpopulations with mean fluorescence intensities and percentage in parent population. The percentage population representing TMRM positivity in (f) actinonin treatment animal group of 6 months latency period and (g) actinonin treatment animal group of 10 months latency period. UT – untreated, AFI – free actinonin i.p and ANI – nanoformulated actinonin i.p. Values are mean ± SE of 3–5 animals. ***p ≤ 0.01.

Figure 8.

Gene expression analysis of lung adenocarcinoma lesions. The egfr mRNA levels at (a) 6 months latency period and (b) 10 months latency period. The folR α mRNA levels at (c) 6 months latency period, (d) 10 months latency period. The pdf mRNA levels at (e) 6 months latency period (f) 10 months latency period. UT – untreated, AFI – free actinonin i.p. and ANI – nanoformulated actinonin i.p. Values are mean ± SE of 3–5 animals *p ≤ 0.05.