Synthesis and anticancer properties of novel dolastatin 10 analogs featuring five-membered heterocyclic rings with a linkable group at the C-terminus.

Dolastatin 10 (Dol-10), a natural marine-source pentapeptide, is a powerful antimitotic agent regarded as one of the most potent anticancer compounds found to date. Dol-10 however, lacks chemical conjugation capabilities, which restricts the feasibility of its application in targeted drug therapy. This limitation has spurred the prospect that chemical structure of the parent molecule might allow conjugation of the derivatives to drug carriers such as antibodies. By first employing docking studies, we designed and prepared a series of novel Dol-10 analogs with a modified C-terminus, preserving high potency of the parent compound while enhancing conjugation capability. The modifications involved the introduction of a methyleneamine functionality at position 4 of the 1,3-thiazole ring, along with the substitution of the thiazole ring with a 1,2,3-triazole moiety, furnished with methylenehydroxy, carboxy, methyleneamine, and N(Me)-methyleneamine tethering functionalities at position 4. Among the synthesized pentapeptides, DA-1 exhibited the highest potency in prostate cancer (PC-3) cells, eliciting apoptosis (IC50 0.2 ± 0.1 nm) and cell cycle arrest at the mitotic stage after at least 6 days of culture. This delayed response suggests the accumulation of cellular stress or significant physiological alterations that profoundly impact the cell cycle. We believe that these novel Dol-10 derivates represent a new and straightforward route for the development of C-terminus modified Dol-10-based microtubule inhibitors, thereby advancing targeted anticancer therapy.

Synthesis and biological evaluation of theranostic Trastuzumab-SN38 conjugate for Near-IR fluorescence imaging and targeted therapy of HER2+ breast cancer.

Here, we report on the design, synthesis, and biological evaluation of a new theranostic antibody drug conjugate (ADC), Cy5-Ab-SS-SN38, that consists of the HER2-specific antibody trastuzumab (Ab) connected to the near infrared (NIR) pentamethine cyanine dye Cy5 and SN38, which is a bioactive metabolite of the anticancer drug irinotecan. SN38 is bound to an antibody through a glutathione-responsive self-immolative disulfide carbamate linker. For the first time, we explored this linker in ADC and found that it to reduce the drug release rate, which is important for safe drug delivery. The developed ADC exhibited specific accumulation and nanomolar anti-breast cancer activity on HER2-positive (HER2+) cell lines but no effect on HER2-. Animals treated with this ADC exhibited good tolerance. In vivo studies have shown that the ADC had good targeting ability for HER2+ tumors with much higher anticancer potency than trastuzumab itself or a mixture of trastuzumab with SN38. Side-by-side HER2+/HER2-xenograft at the 10 mg/kg dose exhibited specific accumulation and reduction of HER2+ tumor but not accumulation or growth inhibition of HER2-counterpart. The self-immolative disulfide linker implemented in this study was proven to be successful, broadening its utilization with other antibodies for targeted anticancer therapy in general. We believe that the theranostic ADCs comprising the glutathione-responsive self-immolative disulfide carbamate linker are applicable for the treatment and fluorescent monitoring of malignancies and anticancer drug delivery.

Effect of Solubilizing Group on the Antibacterial Activity of Heptamethine Cyanine Photosensitizers.

Antibiotic resistance of pathogenic bacteria dictates the development of novel treatment modalities such as antimicrobial photodynamic therapy (APDT) utilizing organic dyes termed photosensitizers that exhibit a high cytotoxicity upon light irradiation. Most of the clinically approved photosensitizers are porphyrins that are poorly excitable in the therapeutic near-IR spectral range. In contrast, cyanine dyes function well in the near-IR region, but their phototoxicity, in general, is very low. The introduction of iodine atoms in the cyanine molecules was recently demonstrated to greatly increase their phototoxicity. Herein, we synthesized a series of the new iodinated heptamethine cyanine dyes (ICy7) containing various solubilizing moieties, i.e., negatively charged carboxylic (ICy7COOH) and sulfonic (ICy7SO3H) groups, positively charged triphenylphosphonium (ICy7PPh3), triethylammonium (ICy7NEt3) and amino (ICy7NH2) groups, and neutral amide (ICy7CONHPr) group. The effect of these substituents on the photodynamic eradication of Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens was studied. Cyanine dyes containing the amide and triphenylphosphonium groups were found to be the most efficient for eradication of the investigated bacteria. These dyes are effective at low concentrations of 0.05 µM (33 J/cm2) for S. aureus, 50 µM (200 J/cm2) for E. coli, and 5 µM (100 J/cm2) for P. aeruginosa and considered, therefore, promising photosensitizers for APDT applications. The innovation of the new photosensitizers consisted of a combination of the heavy-atom effect that increases singlet oxygen generation with the solubilizing group's effect improving cell uptake, and with effective near-IR excitation. Such a combination helped to noticeably increase the APDT efficacy and should pave the way for the development of more advanced photosensitizers for clinical use.

Novel Cyclic Peptides for Targeting EGFR and EGRvIII Mutation for Drug Delivery.

The epidermal growth factor-epidermal growth factor receptor (EGF-EGFR) pathway has become the main focus of selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, namely monoclonal antibodies and small molecule kinase inhibitors. Despite an initial good response rate to these drugs, most patients develop drug resistance. Therefore, new treatment approaches are needed. In this work, we aimed to find a new EGFR-specific, short cyclic peptide, which could be used for targeted drug delivery. Phage display peptide technology and biopanning were applied to three EGFR expressing cells, including cells expressing the EGFRvIII mutation. DNA from the internalized phage was extracted and the peptide inserts were sequenced using next-generation sequencing (NGS). Eleven peptides were selected for further investigation using binding, internalization, and competition assays, and the results were confirmed by confocal microscopy and peptide docking. Among these eleven peptides, seven showed specific and selective binding and internalization into EGFR positive (EGFR+ve) cells, with two of them-P6 and P9-also demonstrating high specificity for non-small cell lung cancer (NSCLC) and glioblastoma cells, respectively. These peptides were chemically conjugated to camptothecin (CPT). The conjugates were more cytotoxic to EGFR+ve cells than free CPT. Our results describe a novel cyclic peptide, which can be used for targeted drug delivery to cells overexpressing the EGFR and EGFRvIII mutation.

Antibody guided activatable NIR photosensitizing system for fluorescently monitored photodynamic therapy with reduced side effects.

Photodynamic therapy (PDT) utilizing an organic dye (photosensitizer) capable of killing cancer cells in the body upon light irradiation is one of the promising non-invasive treatment modalities for many cancers. A known drawback of PDT is a side-effect caused by existing photosensitizers to organs due to insufficient specificity and accidental light exposure of a patient during the delivery of the photosensitizer in the bloodstream. To overcome this issue, we developed a novel antibody guided, activatable photosensitizing system, Ab-mI2XCy-Ac, where the trastuzumab (Ab) is linked to the non-active (not phototoxic and not fluorescent) dye, mI2XCy-Ac, that contains the hydroxyl group protected by acetyl (Ac). This targeting, non-photo-active conjugate was shown to be safely (without detectable side-effects) delivered to the targeted tumor, where it is activated by the esterase-mediated acetyl group cleavage and effectively treats the tumor upon NIR light irradiation. It was demonstrated in the Her2 positive BT-474 tumor mouse model that the treatment efficacy of the activatable photosensitizing system is about the same as for the permanently active photosensitizer, Ab-mI2XCy, while the side-effects are noticeably reduced. In addition, this activatable system enables fluorescence monitoring of the photosensitizer activation events.

A novel, dual action chimera comprising DNA methylating agent and near-IR xanthene-cyanine photosensitizer for combined anticancer therapy.

A facile synthesis, biological evaluation and photodynamic properties of novel activatable anticancer molecular hybrids (chimeras) Ch and I-Ch are described. The chimeras consist of DNA methylating methyl triazene moiety and fluorogenic xanthene-cyanine (XCy) or iodinated xanthene-cyanine (I-XCy) photosensitizer. These two anticancer core structures are bound by means of a self-immolative 4-aminobenzyl alcohol linker. The hydrolytic cleavage of the carbamate protecting group promotes activation of both DNA methylating monomethyl triazene and phototoxic xanthene-cyanine dye providing, in addition, a near-IR emission signal for detection of the drug activation events. Preliminary antiproliferative assay demonstrates that the developed chimeras exhibit higher antitumor activity in the breast cancer cell line upon near-IR light irradiation compared to their structural constituents, xanthene-cyanine photosensitizer and monomethyl triazene substance.

[THERANOSTIC TARGETED DRUG DELIVERY - NEW INSIGHTS INTO DIAGNOSIS AND TREATMENT OF CANCER].

INTRODUCTION: Monoclonal antibodies (mAbs) have become a novel therapeutic modality against cancer due to their specificity, affinity and serum stability. These incredible tools capable of linking variable payload, generate antibody drug conjugates (ADCs) that preserve targeting properties and therefore, specifically deliver drugs to the cancer cells. The drugs should exhibit sub-nano to picomolar cytotoxicity usually affecting fundamental oncogenic mechanisms via mitotic inhibition, DNA alkylation and topoisomerase poisoning. The linkers play a pivotal role in ADC design as they link the cytotoxic payload to the antibody. Linkers maintain the stability of the ADCs in the blood circulation securing their delivery to the cancer cell intact but cleaved when internalized to release the payload. Recently, a more advanced version of ADC, namely theranostic (therapeutic and diagnostic) ADC has emerged. These conjugates allow visualization of targeted drug delivery (TDD) vastly impacting the therapeutic index in cancer treatment. Among diagnostic probes, NIR fluorescent dyes provide important real-time information on the efficacy of drug delivery. A two-dye system comprising one switchable and one constantly light emitting cyanine (Cy) linkers provides the ratiometric quantification of the drug released in the tumor. Due to the enormous repertoire of mAbs, the theranostic ADCs can change the future of cancer diagnosis and treatment.

Discovery of Dolastatinol: A Synthetic Analog of Dolastatin 10 and Low Nanomolar Inhibitor of Tubulin Polymerization.

We developed a highly potent anticancer agent, dolastatinol, which is a methylene hydroxyl derivative of dolastatin 10. Dolastatinol is a synthetic analog of dolastatin 10, synthesized by a solid-phase peptide Fmoc chemistry protocol on 2-chlorotrityl chloride resin utilizing a pH-triggering self-immolative monosuccinate linker. The introduction of the C-terminus hydroxyl methylene functionality preserves the anticancer properties of the parent dolastatin 10, including strong suppression of the cell proliferation, migration, high cytotoxicity. Our research establishes a new facile route toward the further development of C-terminus-modified dolastatin-10-based microtubule inhibitors for anticancer treatment.

Anti-Cancer Effects of Cyclic Peptide ALOS4 in a Human Melanoma Mouse Model.

We examined the effects of ALOS4, a cyclic peptide discovered previously by phage library selection against integrin αvß3, on a human melanoma (A375) xenograft model to determine its abilities as a potential anti-cancer agent. We found that ALOS4 promoted healthy weight gain in A375-engrafted nude mice and reduced melanoma tumor mass and volume. Despite these positive changes, examination of the tumor tissue did not indicate any significant effects on proliferation, mitotic index, tissue vascularization, or reduction of αSMA or Ki-67 tumor markers. Modulation in overall expression of critical downstream αvß3 integrin factors, such as FAK and Src, as well as reductions in gene expression of c-Fos and c-Jun transcription factors, indirectly confirmed our suspicions that ALOS4 is likely acting through an integrin-mediated pathway. Further, we found no overt formulation issues with ALOS4 regarding interaction with standard inert laboratory materials (polypropylene, borosilicate glass) or with pH and temperature stability under prolonged storage. Collectively, ALOS4 appears to be safe, chemically stable, and produces anti-cancer effects in a human xenograft model of melanoma. We believe these results suggest a role for ALOS4 in an integrin-mediated pathway in exerting its anti-cancer effects possibly through immune response modulation.

Targeted methylation facilitates DNA double strand breaks and enhances cancer suppression: A DNA intercalating/methylating dual-action chimera Amonafidazene.

A DNA intercalating agent Amonafide interferes with topoisomerase 2 (Topo II) activity and prevents re-ligation of DNA strands, leading to double strand breaks (DSB). If DSB repair fails, cells stop dividing and eventually die. In a search of approaches to enhance anti-cancer activities of Topo II inhibitors, we hypothesized that introduction of additional damage in proximity to the DSB may suppress DNA repair and enhance cancer cell killing. Accordingly, chimeric molecules were created that target a DNA alkylating component to the proximity of Topo II-induced DSBs. These chimeras consist of Amonafide or its 4-amino isomer, and DNA methylating methyl triazene moiety Azene protected with a carbamate group, connected via linker. Treatment of cancer cells with the chimeric molecules leads to significantly higher number of DSBs, which were repaired slower compared to Amonafide or monomethyl triazene-treated cells. On the other hand, methyl triazene linked to non-intercalating Amonafide analogs was ineffective. Together, these data strongly support our hypothesis. In line with increased DSBs, the chimeric molecules exhibited significantly higher antiproliferative activity in cancer cell lines compared to Amonafide or monomethyl triazene constituent Azene. We utilized the fluorescent properties of chimera Amonafidazene to develop ''photo-switchable'' reporting system to monitor the prodrug activation. Using this approach, we found that the chimera accumulated and was activated at the tumor sites specifically and demonstrated significantly stronger tumor suppressing activities compared to Amonafide in a xenograft model. Therefore, targeting alkylating groups to the proximity of DSB sites may become an effective approach towards enhancing anti-cancer activities of inhibitors of topoisomerases.

Ratiometric Fluorescence Monitoring of Antibody-Guided Drug Delivery to Cancer Cells.

Ratiometric measurements utilizing two independent fluorescence signals from a dual-dye molecular system help to improve the detection sensitivity and quantification of many analytical, bioanalytical, and pharmaceutical assays, including drug delivery monitoring. Nevertheless, these dual-dye conjugates have never been utilized for ratiometric monitoring of antibody (Ab)-guided targeted drug delivery (TDD). Here, we report for the first time on the new, dual-dye TDD system, Cy5s-Ab-Flu-Aza, comprising the switchable fluorescein-based dye (Flu) linked to the anticancer drug azatoxin (Aza), reference pentamethine cyanine dye (Cy5s), and Her2-specific humanized monoclonal Trastuzumab (Herceptin) antibody. The ability of ratiometric fluorescence monitoring of drug release was demonstrated with this model system in vitro in the example of the human breast cancer SKBR3 cell line overexpressing Her2 receptors. The proposed approach for designing ratiometric, antibody-guided TDD systems, where a "drug-switchable dye" conjugate and a reference dye are independently linked to an antibody, can be expanded to other drugs, dyes, and antibodies. Replacement of the green-emitting dye Flu, which was found not detectable in vivo, with a longer-wavelength (red or near-IR) switchable fluorophore should enable quantification of drug release in the body.

Quantification of Drug Release Degree In Vivo Using Antibody-Guided, Dual-NIR-Dye Ratiometric System.

Fluorescent dyes linked to drug delivery systems provide important real-time information on the efficacy of drug delivery. However, the quantitative monitoring of drug distribution is challenging because of interferences from the biological sample and instrumental setup. To improve quantification of anticancer drug delivery followed by drug release in tumor, we equipped an antibody-drug conjugate (ADC) with a turn-on near-infrared (NIR) dye, sensitive to drug release, and a reference NIR dye. In this study, chlorambucil (CLB) was chosen as a model anticancer drug and Trastuzumab monoclonal antibody specific to Her2 receptors overexpressed in many tumors was taken as the carrier. The advantage of the obtained dual-dye ratiometric system for drug release monitoring was demonstrated in mice model.

Drug Combination in Cancer Treatment-From Cocktails to Conjugated Combinations.

It is well recognized today that anticancer drugs often are most effective when used in combination. However, the establishment of chemotherapy as key modality in clinical oncology began with sporadic discoveries of chemicals that showed antiproliferative properties and which as a first attempt were used as single agents. In this review we describe the development of chemotherapy from its origins as a single drug treatment with cytotoxic agents to polydrug therapy that includes targeted drugs. We discuss the limitations of the first chemotherapeutic drugs as a motivation for the establishment of combined drug treatment as standard practice in spite of concerns about frequent severe, dose limiting toxicities. Next, we introduce the development of targeted treatment as a concept for advancement within the broader field of small-molecule drug combination therapy in cancer and its accelerating progress that was boosted by recent scientific and technological progresses. Finally, we describe an alternative strategy of drug combinations using drug-conjugates for selective delivery of cytotoxic drugs to tumor cells that potentiates future improvement of drug combinations in cancer treatment. Overall, in this review we outline the development of chemotherapy from a pharmacological perspective, from its early stages to modern concepts of using targeted therapies for combinational treatment.

Expedient synthesis and anticancer evaluation of dual-action 9-anilinoacridine methyl triazene chimeras.

The efficient synthesis of molecular hybrids including a DNA-intercalating 9-anilinoacridine (9-AnA) core and a methyl triazene DNA-methylating moiety is described. Nucleophilic aromatic substitution (SN Ar) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low micromolar IC50 values in antiproliferative assays performed with various cancer cell lines. In comparison, compound 7b outperformed DNA-intercalating drugs like amsacrine and AHMA. Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA-repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by intercalation of the acridine core.

Photodynamic effect of novel hexa-iodinated quinono-cyanine dye on Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus (S. aureus) is a Gram-positive bacteria and major human pathogen which can cause a wide variety of serious infections when it enters the bloodstream or internal tissues. Antimicrobial photodynamic therapy (APDT) utilizing a light-activated dye (photosensitizer) is a powerful method for in vitro and in vivo eradication of S. aureus and other pathogenic bacteria. However, the development of highly efficient, long-wavelength photosensitizers showing high phototoxicity to pathogens and low dark toxicity is still challenging. AIM: To develop a highly efficient, long-wavelength photosensitizer for photodynamic inactivation of S. aureus. METHOD: Synthesis of the new photosensitizer, hexa-iodinated quinono-cyanine dye IQCy and investigation of the dark and light-induced toxicity of this dye compared to known photosensitizers Chlorin e6 (Ce6) and HITC towards S. aureus. RESULTS: When exposed to 14.9 J/cm2 white LED light, 0.5 µM of IQCy, Ce6 and HITC inactivate, respectively, 99 %, 40 % and 30 % of S. aureus and at 0.05 µM and 27.9 J/cm2 - 71 %, 18 % and 9%, which is much better compared to Ce6 and HITC. IQCy exhibits no dark toxicity at least at 10 µM dye concentration. CONCLUSIONS: IQCy demonstrates a more pronounced photodynamic inactivation of S. aureus as compared to Ce6 and HITC and can be employed for the eradication of these bacteria at lower concentration and reduced light dose.

Peptide-Driven Targeted Drug-Delivery System Comprising Turn-On Near-Infrared Fluorescent Xanthene-Cyanine Reporter for Real-Time Monitoring of Drug Release.

Targeted drug delivery (TDD) is an efficient strategy for cancer treatment. However, the real-time monitoring of drug delivery is still challenging because of a pronounced lack of TDD systems capable of providing a near-infrared (NIR) fluorescence signal for the detection of drug-release events. Herein, a new TDD system, comprising a turn-on NIR fluorescent reporter attached to an anticancer drug and targeting peptide, is reported. This system provides both TDD and NIR fluorescence monitoring of drug-release events in target tissue. In this TDD system, a new carboxy-derivatized xanthene-cyanine (XCy) dye is attached to an anticancer drug, chlorambucil (CLB), through a hydrolytically cleavable ester linker and coupled to a targeting peptide, octreotide amide (OCTA), which is specific to somatostatin receptors SSTR-2 and STTR-5 overexpressed on many tumor cells. This OCTA-G-XCy-CLB (G: γ-aminobutyric acid) conjugate exhibits no detectable fluorescence, whereas, upon the hydrolytic cleavage of the ester linker, a bright NIR fluorescence appears at λ≈710 nm; this signals release of the drug. Real-time TDD monitoring is demonstrated for the example of the human pancreatic cancer cell line overexpressing SSTR-2 and STTR-5, in comparison with the noncancerous Chinese hamster ovary cell line, which contains a reduced number of these receptors.

Dark Antibacterial Activity of Rose Bengal.

The global spread of bacterial resistance to antibiotics promotes a search for alternative approaches to eradication of pathogenic bacteria. One alternative is using photosensitizers for inhibition of Gram-positive and Gram-negative bacteria under illumination. Due to low penetration of visible light into tissues, applications of photosensitizers are currently limited to treatment of superficial local infections. Excitation of photosensitizers in the dark can be applied to overcome this problem. In the present work, dark antibacterial activity of the photosensitizer Rose Bengal alone and in combination with antibiotics was studied. The minimum inhibitory concentrations (MIC) value of Rose Bengal against S. aureus dropped in the presence of sub-MIC concentrations of ciprofloxacin, levofloxacin, methicillin, and gentamicin. Free Rose Bengal at sub-MIC concentrations can be excited in the dark by ultrasound at 38 kHz. Rose Bengal immobilized onto silicon showed good antibacterial activity in the dark under ultrasonic activation, probably because of Rose Bengal leaching from the polymer during the treatment. Exposure of bacteria to Rose Bengal in the dark under irradiation by electromagnetic radio frequency waves in the 9 to 12 GHz range caused a decrease in the bacterial concentration, presumably due to resonant absorption of electromagnetic energy, its transformation into heat and subsequent excitation of Rose Bengal.

Data on peptidyl platform-based anticancer drug synthesis and triton-x-based micellar clusters (MCs) self-assembly peculiarities for enhanced solubilization, encapsulation of hydrophobic compounds and their interaction with HeLa cells.

The data presented here refer to a research article entitled "Self-Assembled Micellar Clusters Based on Triton-X-family Surfactants for Enhanced Solubilization, Encapsulation, Proteins Permeability Control, and Anticancer Drug Delivery" Solomonov et al., 2019. The present article provides the General Procedure for clusterization of Triton-X-based micelles and the effect of (i) metal ion, surfactant, and chelator concentration on the developed clusters formation, (ii) surfactant-chelator relation change, (iii) metal ion-micelles concertation ratio variation, (iv) metal ion replacement, (v) solvent replacement, (vi) kinetics of clusters formation, (vii) hydrophobic fluorescent dye (Coumarin 6) solubilization in aqueous MCs media, (viii) novel anticancer peptidyl drug synthesis and characterization and (ix) the viability of HeLa cells with and without the presence of drug-free Triton-X-based family MCs. These data provide additional insights useful for understanding all aspects of the micellar clusters formation, optimization, and control.

Self-assembled micellar clusters based on Triton-X-family surfactants for enhanced solubilization, encapsulation, proteins permeability control, and anticancer drug delivery.

Non-ionic surfactants have raised a considerable interest for solubilization, encapsulation, permeabilization and controlled release of various compounds due to their unique physicochemical properties. Nevertheless, it is still challenging to create convenient self-assembled multifunctional materials with high solubilization and encapsulation capacities by preserving their advanced capabilities to protect loaded cargos without altering their characteristics. In this work, we present an extended concept of micellar clusters (MCs) formation based on partial entrapment and stabilization of chelate ligands by hydrophobic forces found on the non-ionic surfactant micelle interface of the Triton-X family (TX-100/TX-114), followed by subsequent complexation of the preformed structures either by metal ions or a supporting chelator. The formation aspects, inner structure and the role of external factors such as the addition of competitive ligands have been extensively studied. MCs loaded by hydrophobic fluorescent compounds with high encapsulation efficiency demonstrate an excellent optical response in aqueous media without crystallization as well as sufficient increase in solubility of toxic hydrophobic compounds such as bilirubin (>50 times compared to pure surfactants). Furthermore, Triton-X-based MCs provide a unique feature of selective permeability to hydrophilic ligand-switching proteins such as UnaG and BSA demonstrating bright "turn-on" fluorescence signal either inside the cluster or on its interface via complexation. The proposed strategies allowed us to successfully encapsulate and visualize a newly synthesized, highly hydrophobic anticancer PTR-58-CLB-CAMP peptide drug, while MCs loaded by the drug exhibit a considerable antitumor activity against HeLa cells.

Theranostic system for ratiometric fluorescence monitoring of peptide-guided targeted drug delivery.

Conjugation of an anticancer drug with a cancer-specific carrier and a fluorescent dye to form a theranostic system enables real time monitoring of targeted drug delivery (TDD). However, the fluorescence signal from the dye is affected by the light absorption and scattering in the body, photobleaching, and instrumental parameters. Ratiometric measurements utilizing two fluorescence signals of different wavelengths are known to improve sensitivity, reliability and quantitation of fluorescence measurements in biological media. Herein, a novel theranostic system comprising the anticancer drug chlorambucil (CLB), cancer-specific peptide octreotide amide (OctA), and a long-wavelength dual fluorescent cyanine dye IRD enabling ratiometric monitoring of drug delivery was developed and evaluated on the cancer cell line PANC-1.