Iodine loaded nanoparticles with commercial applicability increase survival in mice cancer models with low degree of side effects

Abstract Background The recorded use of iodine in medicine, dates to 5000 BC. Molecular iodine (I2) has been claimed to exert an antineoplastic effect that triggers apoptotic and re‐differentiation mechanisms in different types of cancer cells in animal studies. Hitherto, all experiments published have been carried out with I2 diluted in water preparations resulting in the administration of ionized iodide, either alone or in combination with low levels of I2. To maximize the levels of I2 by avoiding water solutions we have managed to develop a colloidal nano particle (NP) loaded with I2 with a Z‐average of 7‐23 nm with remarkable stability, preferable osmolality and commercial applicability. Aims Here we report the results from formulation and pre‐clinical studies with the rationale: a) to find a tolerable dose of the I2 NP system delivered intravenously or per‐orally, and b) to determine if the tolerable doses are efficacious in murine models of cancer. Methods and Results A novel drug delivery system with I2 NP was formulated and murine cancer models with CT26, MDA‐MB‐231 and LL/2 cells were used to analyse the efficacy. Despite the formulation challenges we were successful in constructing stable NPs loaded with I2 which have convincing commercial applicability. We conclude that administration of the NP I2 drug delivery system: 1. Blunted tumour growth in a xenograft breast cancer model; 2. Had a significant effect on survival in the orthotopic, syngeneic lung metastasis model; 3. Showed reduced tumour burden in post‐mortem evaluation and; 4. Was associated with low degree of side effects. Conclusions Taken all together, our findings indicate that the NP I2 drug delivery system may serve as a novel effective cancer treatment with low degree of side effects. This is something which needs further exploration including confirmation in future clinical trials.


| INTRODUCTION
The recorded use of iodine in medicine, dates to 5000 BC when seaweed and sea sponges were used to shrink goitres. Chemically, the term iodine represents any form of the molecule and includes molecular iodine (I 2 ), iodide salts (NaI or KI), iodate (NaIO) in addition to lipids or proteins containing iodine such as iodotyrosine or iodolactones.
Iodide salts have been studied extensively and are often used in supplementation to entire populations.
Iodine is important in thyroid hormone formation and function in addition to independent physiological effects. Iodine also acts intracellularly as an antioxidant, anti-inflammatory and proapoptotic agent essential for health and cellular renewal. 1 Molecular iodine (I 2 ) has been claimed to exert an antineoplastic effect that triggers apoptotic and re-differentiation mechanisms in different types of cancer cells. 2 Moreover, I 2 may also serve as an immune modulator. It has been reported that several immune cell types can internalise iodine, which depending on the cellular context, imply antibacterial and anti-inflammatory effects, or induction of immune response. 3,4 It is well known that I 2 supplementation at millimolar concentrations exert beneficial effects in women with mammary fibrocystic disease or mastalgia, without negative effect on their thyroid function or general health. Moreover, it has been demonstrated that such anti-proliferative and anti-inflammatory effects can also be seen in models of benign prostatic hyperplasia and cancer in various organs that absorb iodine. 2 Recent findings show that I 2 exerts synergistic effects when used as an adjuvant to Doxorubicin antineoplastic treatments in animal models. 5 Hitherto, all experiments published have been carried out with I 2 or ions thereof, diluted in water as the only means of formulation and administration per orally. Such iodine solutions mainly contain iodide, such as potassium iodide whereas the ideal formulation would contain predominantly I 2 . Hence, it is questionable if previous published studies with water solutions of iodine have had larger proportions of I 2 .
With this lacuna as a basis, we have managed to develop a colloidal nano particle (NP) loaded with I 2 with a Z-average of 7-23 nm with remarkable stability and preferable osmolality with the aim to specifically target and inhibit the growth of cancer cells. There is a high proportion of bound I 2 and product stability exceeds 6 months of shelf life in room temperature. Manufacturing to the highest standard has been executed by one of Europe's largest commercial manufacturers of extemporaneous medicines.
Here we report the results from formulation and pre-clinical studies with two main objectives: (a) to find a tolerable dose of the I 2 NP system delivered intravenously or per-orally, and (b) to determine if the tolerable doses are efficacious in murine models of cancer.

| Animals
Female BALB/c, C57BL/6J or athymic nude mice (6-8 weeks of age) were acquired from Charles River (Germany). They were housed in groups of 3 to 8 mice in plastic cages with adsorbent bedding in temperature and light-dark cycle (12:12 h) controlled rooms. Food and water were available ad libitum. Animals were left to acclimatise for at least 5 days before the start of experimental procedures. it is known for its good solubilising properties and the ability to spontaneously form micelles. Different concentrations (10%-30% (w/w)) of KEL were evaluated and the concentration found suitable was 10% (w/w). Different concentrations of iodine, 0.4%-5% (w/w) were evaluated and the suitable concentration was found to be 0.4% (w/w).
Formulations containing higher amounts of iodine did separate during storage resulting in a brownish milk-like preparation or precipitated.
Chemicals used for manufacture is described in Table 1  The inherent pH of the formulations is about 2 and the osmolality is hypotonic of around 42 mOsm/kg (data not shown). As seen in Table 3, the micelle size distribution is found to be 7-11 nm (Z-average) for CIP1 formulations and the corresponding placebos. No effect of pH on micelle size distribution can be seen even when the pH was adjusted using 5 M sodium hydroxide. The micelle size growth of CIP1 was temperature dependent and phase separation occurs after 3 months for samples stored at 25 C/60% RH and after 12 months for samples stored at 5 C/Ambient RH. The amount of total iodine was 0.40%-0.43% (w/w) and the amount of free iodine was 23%-37% (w/w) of the total amount of iodine, meaning that roughly two thirds of iodine is bound to the micelle.
Given the challenges with stability, osmolality and pH, various buffering systems were explored where CIP3 formulated with a citrate buffer to prevent precipitation was successful. The amount of citrate buffer needed was evaluated by titration starting at 100 mM citrate and ending at 140 mM citrate with respect to withstanding change in pH.
The suitable CIP3 formulation evaluated is a dark orange/brownish solution without visible particles. The change in pH after addition of iodine was 0.1 pH unit, and no temperature dependent change was recorded when the samples were stored for the citrate buffer formulation ( Table 4). The 140 mM citrate buffer formulation is slightly hypertonic.
The micelle size distribution is found to be 15-23 nm (z-average) for the active CIP3 formulation. No obvious temperature dependent change in micelle size distribution over time, for the samples analysed 4, 10, 29 and 35 weeks after manufacture, was recorded for either active or placebo formulations. In addition, only minor visual changes were observed over the time period ( Figure 1). An out of trend result for the data presented after 10 weeks for the active formulation can be seen. This as the micelle size distribution appear to get smaller for the samples stored at 25 C/Ambient RH, but when comparing the micelle distribution for the samples stored at 5 C/Ambient RH a consistent micelle size distribution is seen, and the results at 25 C/Ambient RH after 10 weeks are considered out of expectation (

| Cancer models
The syngeneic colon cancer model cells ( All the experiments performed are summarised in Figure 2. T A B L E 3 Concentration of iodine (free and total), micelle size distribution and pH for CIP1. F I G U R E 1 CIP3 -Minor colour difference after 29 weeks between storage at 5 C (right) and 25 C (left).

| Statistical analysis
Data was analysed using GraphPad Prism software (San Diego, CA

| Pharmacokinetics
The pharmacokinetic properties of CIP1 were evaluated following a

| Cancer models
To determine the cancerostatic potential of CIP1, efficacy was evalu-  Whereas per-oral CIP1 at pH 2 to 3 had no effect on tumour development, intravenous CIP1, at both physiological ( p = .024) and F I G U R E 5 Absolute (A) and relative (B) tumour growth of MDA-MB-231 tumours and relative body weight (C) following repeated administration of vehicle or CIP1, via intravenous or per-oral delivery or cisplatin. Relative growth is calculated as percent of volume/body weight on Day 0. Data is presented as mean ± SEM, n = 8. An asterisk indicates a p-value <.05. A tumour with a diameter of <3 mm was considered small; a tumour with a diameter of >3 mm was considered large. In bar graphs, data is presented as mean ± SEM, n = 8 for survival graph; n = 4-8 for tumour burden. An asterisk indicates a p-value <.05. It has been suggested that such iodine formulations with predominantly sodium iodide and sodium iodate lead to the generation of molecular iodine in the stomach upon dissolution in the gastric fluid, 9 something which remains to be proven.
In contrast our unique formulation has a large proportion of I 2 bound to the NPs. I 2 is reportedly the active element of iodine, and therefore NPs loaded with iodine has the potential to exert enhanced cancerostatic effects in a drug delivery fashion compared to previous positive trials with water-based formulations of iodine. 2 Another advantage is that several studies 10,11 have shown I 2 to be less thyrotoxic compared to water soluble iodide which has a different tissue distribution in mammals.

| Tolerability and pharmacokinetics
Intravenous administration of CIP1, at an acidic pH, was tolerable at 2.9 mg/kg. At higher doses, animals presented with local adverse reactions, including development of a swollen, blueish black tail, indicative of inflammation and necrosis. This was likely a reaction to the low pH of the test item, which would have likely negatively affected the integrity of the injected blood vessels. Indeed, when CIP1 was pH-adjusted to a physiological pH, intravenous administration was tolerated at a higher dose of 4 mg/kg.
At a physiological pH, repeated intravenous administration of doses higher than 4 mg/kg also led to local reactions, including swelling, and blackening of the tail. I 2 is a strong oxidising agent, and high concentrations could lead to local irritation and oxidative injury. 12 Per-oral administration of CIP1, at an acidic pH, was tolerable at much higher doses, as compared to intravenous injection. At the highest feasible per-oral dose, 80 mg/kg, no adverse effects were seen.
Interestingly, the pH adjusted CIP1 to a physiological pH reduced the tolerability with a maximum tolerable dose of 26.7 mg/kg. Systemic reactions were observed, including weight loss, piloerection and preterm death. The reason for this is unclear, but it is possible that when administering the pH adjusted CIP1 per-orally, the compound will immediately be exposed to an acidic environment, and the pH adjustment performed ex vivo will be reversed. As a result, the toxic effects seen are unlikely due to the pH of the compound per se, but possibly due to molecular changes of Iodine upon pH adjustment.

| Cancer models
No effect on tumour growth was evident for CIP1 in the CT26 model. Conventional chemotherapy is linked to many well described severe side effects such as acute and chronic toxicity. 13,14 Most organs of the body are affected including vital ones such as the heart, lungs and brain. Moreover, chronic effects of chemotherapy can entail drug resistance, carcinogenicity and infertility. Hence, it is interesting to note that the effect of per oral administration of CIP3 was equivalent to that seen following administration of cisplatin but with absence of toxicity.
Interestingly, ancient traditional medicine systems have long used iodine-rich seaweeds as a cancer treatment and iodine supplementation has been linked to positive reductions in breast and prostate cancer epidemiology. 15 Despite such promising data, no major clinical translation into a pharmaceutical product has been undertaken. However, a recent pilot study has analysed the adjuvant effect of a water solution of iodine together with chemotherapeutic treatment against breast cancer. The preliminary results indicate that the supplementation of iodine, notably iodine ions, improves the effectiveness of the treatment, decreasing side effects and increasing disease-free survival specially in advanced conditions (stage III). They were also able to show that iodine supplementation induces tumour re-differentiation and the reactivation of antitumour immune responses. 16 It is understandable that without an effective and commercially available pharmacological product aligned with standard operation Initially all experiments were carried out with CIP1 but due to problems of stability and pH adjustments we added citrate as a buffering agent. This improved the shelf-life stability considerably of the formulation and omitted the need for pH adjustments. In the followup study, that was performed to validate the findings of CIP1 delivered at physiological pH, we included the new buffered formulation of CIP; CIP3. Here we were able to demonstrate equivalent efficacy.
However, in future experiments we will consider including a full tolerability assessment of CIP3 to determine whether higher doses of this formulation can be delivered without inducing toxic effects. In addition, the effect of CIP3 in both colon and breast cancer models may also be repeated.

| CONCLUSION
Despite the formulation challenges, we were successful in constructing stable nano particles (NPs) loaded with I 2 , which have convincing commercial applicability. Taking in consideration the methodological constraints of this study we can conclude that administration of the NP I 2 drug delivery system:

FUNDING INFORMATION
This work was financed by Shigeru AG, Gerbergasse 48 CH-4001 Basel, Switzerland.

CONFLICT OF INTEREST STATEMENT
Colloidal Iodine is being developed by Shigeru AG which financed the research and development performed by Adlego/Scantox and APL without any demands of retribution

DATA AVAILABILITY STATEMENT
Data is available upon request.

ETHICS STATEMENT
All appropriate ethical approvals were sought and approved.