*3.3. Drug Loading into Dendrimersomes*

To explore the potential of dendrimersomes as drug carriers, we encapsulated three anti-tumor drugs—doxorubicin, methotrexate and 5-fluorouracil into dendrimersomes. Concentrated solutions of a drug and a dendron were rapidly mixed in water, sonicated and incubated for 48 h, followed by removal of non-encapsulated drug by dialysis. This procedure yields drug-loaded dendrimersomes with low size dispersion (PDI < 0.3) with a high degree of drug encapsulation (ca. 65% for doxorubicin and 75% for methotrexate) (Table 1). On the contrary, 5-fluorouracil encapsulated much less efficiently: for ca. 25%. The differences in the drug encapsulation efficiency resulted in 10-fold variation of the drug loading capacity: ~20% for doxorubicin and methotrexate vs. ~2% for 5-fluorouracil (Table 1). Such a dissimilarity is likely explained by the hydrophobicity of the encapsulated compound: doxorubicin and methotrexate can not only be entrapped into the inner cavity of a dendrimersomes but also incorporated into a bilayer (that can be observed in the TEM images (Figure 4) by the thickening of dendrimersomes' membranes), whereas for 5-fluorouracil, the entrapment likely prevails. Thus, the efficiency of 5-fluorouracil encapsulation can give an idea of the efficiency of the physical entrapment of compounds into the hydrophilic interior of dendrimersomes. It should be noted that methotrexate appeared to be additionally retained on the dendrimersomes' surface due to electrostatic interactions, as suggested by zeta potential values and TEM images. Drug-loaded dendrimersomes have mean diameter less than 100 nm that makes them suitable for drug delivery studies (Table 1).


**Table 1.** Properties of drug-loaded dendrimersomes.

<sup>1</sup> DS-dendrimersomes; <sup>2</sup> Mean ± S.D.; <sup>3</sup> rough estimation, see Section 2.8.

**Figure 4.** Representative TEM images of doxorubicin-loaded (DOX@DS), methotrexate-loaded (MTX@DS) and 5-fluorouracil-loaded (5FU@DS) dendrimersomes (top) and cartoons showing tentative drug distribution in dendrimersomes (bottom). Scale bar represents 50 nm. Black arrows show the thickness of a bilayer.

We have studied the pH dependence of the drug release from dendrimersomes using doxorubicin-loaded constructions as a model. The release was followed by measuring fluorescence intensity of dialysate (see Section 2.9). The rate of the drug release from dendrimersomes has not been found to increase significantly in slightly acidic medium (Figure S1): The half-release time being 6.3 ± 1.4 h at the pH 7.0 decreased to 6.0 ± 1.5 h (pH 6.5) and 5.4 ± 1.4 h (pH 6.0). However, overall fluorescence intensity of dialysate was higher with the decrease of the pH. The completeness of the doxorubicin release from dendrimersomes after 24 h was >80%. Thus, the pH-dependent behaviour shown for blank dendrimersomes (Figure 3) likely takes place in drug-loaded constructions as well, triggering the release of a drug in slightly acidic media frequently occurring in endocytic vesicles at early stages of endosome maturation [35].
