(701g) Stimuli Responsive Lipid Nanotubes for Drug and Protein Delivery

Lipid nanotubes (LNT) are one of the most attention drawing systems due to their superiorities provided by their asymmetric geometry such as high interior volume, high drug loading capacity, easy surface modifications and high circulation persistence as well as their high biocompatibility. Thus, they have high potential to be used in many different applications ranging from electronics to bionanotechnology. One of the most important applications of LNTs is their usage as drug delivery agents. Due to their exceptionally high interior volume compared with most of the other carrier systems they are expected to effectively entrap both small drugs and biomacromolecules.

Recently, we designed a molecule called as AQUA (AQ-NH-(CH₂)₁₀COOH, where AQ is anthraquinone group) that can form self-assembled lipid nanotubes. The molecular structure has been designed very carefully so that each group in the structure has key properties for both nanotube formation and its functions. AQUA LNTs have dual responsive character provided by COOH and AQ groups and its morphology can be controlled reversibly by external stimuli (pH and redox reaction). In the design step of AQUA molecules, we especially took into consideration to use them as delivery agents for some selected pharmaceuticals such as Doxorubicin (DOX), Hemoglobin (Hb) and BSA.

Total Hb entrapment capacity is determined by UV-vis spectroscopy from the absorbance value of the samples which were treated with Drabkinâ??s reagent at 420 nm. The entrapment capacity is significantly affected by the solution pH and the loaded Hb amounts are constant and at its maximum, in the pH range of 5.0-7.0. AQUA nanotubes can hold Hb approximately two times that of their own weight. (i.e loading ratio is ~2) However, loading ratio decreases to 1.1 as the pH is increased to 9.0 due to the reduced electrostatic attraction. However, even at the high pH values AQUA nanotubes can carry significant amount of Hb. The capability to make Ï?-Ï? stacking interactions of the AQ group in the AQUA increase the loading capacity and helps to obtain high loading ratios in a wide pH range.

Hb release profile to PBS buffer at pH 7.0 changes with loading pH and release temperature however, even at the maximum measured release conditions, AQUA nanotubes keep more than 80 % of the initially loaded Hb after three days and from this time the Hb release almost vanish. This such a low Hb release at the physiological conditions along with the high loading capacity, is very important and advantageous for the artificial blood application potential of AQUA nanotubes.

In the BSA loading and release studies Biuret method is used and the BSA amount is determined from absorbance value at 540 nm by UV-vis spectroscopy. Maximum BSA loading is achieved at pH 3.0 and pH 6.0 with the loading ratio of 0.8. When the BSA loading ratios of AQUA nanotubes are compared with the systems in the literature, it is seen that AQUA nanotubes are loaded with 10-50 times more BSA than most of them systems in the literature. At room temperature, the highest release is observed at pH 7.0 and totally 59 % ±0.5 of the loaded BSA is released after 48 hours which is determined as 18 %±0.5 for pH 5.0 and 20 %±0.5 for pH 6.0. When the temperature is increased to 37 ^oC at pH 7.0, the total released amount in the 48±0.5 hours increases to 68 %±0.5 due to the increase of the water solubility of BSA with temperature [1]. Unlikely to Hb, high release rates can be reached for BSA. The in vitro release profiles from AQua nanotubes shows opposite behaviors for Hb and BSA and both of those profiles are advantageous for different purposes. The very low release percentages for the Hb even when it is at its maximum, is a desired result for the artificial blood applications while the very high and pH and temperature dependent release profiles for BSA are pleasing for the controlled release applications.

The entrapment and release studies carried out with two different model proteins showed that the special functional groups of the AQUA molecule are quite effective to obtain strong attraction interactions with the guest molecules and it is possible to control these interactions on demand with the help of the environmental triggers due to the stimuli responsive character of our nanotubes. After these promising results the entrapment and release studies of the small drugs was carried out by using doxorubicin (DOX) as the model drug.

DOX is an anthraquinone based antibiotic which is commonly used for cancer treatment. However, it has various important side effects such as cardiotoxicity, supressing blood cell production, low therapeutic index, etc. So, developing effective carrier systems for DOX is a widely interested topic [2]. One of the most common cancer types for which DOX is used as the therapeutic agent is the colorectal cancer (CRC). Although parenteral ways are generally preferred for the treatment of the colorectal cancer, oral or intracolonic administrations can also be used.

AQUA and DOX have some similarities in their molecular structure and they both contain anthraquinone groups which we believe affects the affinity of DOX to AQUA LNTs via Ï?-Ï? stacking interactions. In addition, the presence of carboxylic acid group in the molecular structure of AQUA helps us to encapsulate significant amount of DOX due to the hydrogen bonding and electrostatic interactions. There are some studies in the literature in which DOX is added to molecule structure/carrier via a covalent bond. These kinds of approaches will require more complex synthesis procedures which will prevent their use in large scale applications. Here we present a drug delivery system formed by relatively simple, but smartly tailored molecules.

The pH sensitive character of the AQUA LNTs is advantageous for increasing their efficiency on drug entrapment and release as well as providing selective release of the DOX to the tumor cells. DOX loading capacity is effected by the parameters such as drug concentration, pH, drug to carrier mass ratio. The maximum loading capacity is reached at pH 9 when the DOX/LNT ratio is 1/1 (g/g). In this condition AQUA LNTs can entrap almost as much DOX as their own mass. This loading capacity is significantly higher than the ones that could be reached in the literature [3]. Also it has been found that the release of DOX from the AQUA LNTs changes depending on the temperature and pH. The AQUA LNTs releases significantly more DOX at pH 5.5 which is the pH of the many cancer tissues than physiological pH (i.e. 7.0) This shows that AQUA LNTs selectively releases the DOX to the cancer tissue which is very advantageous for their usage as the carrier system in the cancer treatment.

The abovementioned results show that the rationally designed new stimuli responsive nanotube system is versatile as a highly functional carrier system for various different guest molecules changing from small drugs to biomacromolecules.

 [1] M. A. Masuelli, Advances in Physical Chemistry, 2013, 2013, 360239

 [2] B.D. Weinberg, H. Ai, E. Blanco, J.M. Anderson, J. Gao, J Biomed Mater Res., 2007, 81, 161.

 [3] S. Ilbasmis-Tamer, H. Unsal, F. Tugcu-Demiroz, G.D. Kalaycioglu, I.T. Degim, N. Aydogan, Colloids Surf B Biointerfaces, 2016, 143, 406.