Reversible glycosidic switch for secure delivery of molecular nanocargos

Therapeutic drugs can leak from nanocarriers before reaching their cellular targets. Here we describe the concept of a chemical switch which responds to environmental conditions to alternate between a lipid-soluble state for efficient cargo loading and a water-soluble state for stable retention of cargos inside liposomes. A cue-responsive trigger allows release of the molecular cargo at specific cellular sites. We demonstrate the utility of a specific glycosidic switch for encapsulation of potent anticancer drugs and fluorescent compounds. Stable retention of drugs in liposomes allowed generation of high tumor/blood ratios of parental drug in tumors after enzymatic hydrolysis of the glycosidic switch in the lysosomes of cancer cells. Glycosidic switch liposomes could cure mice bearing human breast cancer tumors without significant weight loss. The chemical switch represents a general method to load and retain cargos inside liposomes, thereby offering new perspectives in engineering safe and effective liposomes for therapy and imaging.

Remarks to the Author: The manuscript has intriguing approach of hydrophilic-lipophilic switch enabled loading of anticancer drugs in the liposome aqueous core. Extensive evaluation of the formulation has been carried out to establish the proof of concept. Authors' have also addressed the previous reviewer's comments in the revised manuscript. However, there are following comments that need to be resolved before publication. 1. C amptothecins have been noted to be very unstable in aqueous media due to their closed lactone ring which opens at alkaline pH (pH>5. 5) forming inactive open lactone form. The rates of hydrolysis of C PT in a PBS (pH 7.4) have been studied and it has been shown that only 10% activity remained after 2 hr in PBS. Moreover, liposome development has been targeted to stabilize the compounds in the bilayer where its lactone is protected from the hydrolytic aqueous phase. Keeping these in mind and use of pH 8.5 inside liposomes, I think authors should describe what actually they are measuring in the liposomes i.e. is it the of AC G closed lactone or AC G open lactone? The explanation makes it confusing, as it could be a mixture of components inside such as 9AC -GW open lactone, 9AC -GW closed lactone, calcium salts of lactone, calcium salt of glucuronate etc.). This in turns requires explanation for different precipitation phenomenon inside the liposomes. 2. Authors showed that the switch molecules are hydrolyzed to water soluble counterparts (Fig 2 C onversion of 9AC -GL to 9AC -GW), however, they never emphasized on the fact that it is not actually the conversion of 9AC -GL to 9AC -GW and rather it is conversion to open lactone 9AC -GW. Authors need to bring this to notice in the manuscript and also explain the disposition occurring in the cancer cells under acidic conditions which is essential for activity of 9AC (about lactone ring). 3. This in turn also warrants the explanation for further disposition and activity of other molecules (glucuronic acid, calcium and benzyl alcohol) generated at equimolar or higher concentrations as drug after the glucuronidase cleavage. Don't these molecules impart their effects on the cytotoxicity or other effects? 4. Secondly, when authors have used methanol for esterification, this makes it important that authors explain the release of methanol upon hydrolysis. As methanol could be toxic to the cells, they need to rationally justify based on the quantity of the methanol that the cells will be exposed upon dosing and give relevant deduction. 5. Authors' generalization to apply the switch methodology to a wide range of compounds is highly speculative. Even though 9AC , BQC and 4MU had similar log P values, their switch versions i.e. W and L versions gave different partitioning to the molecules with logP of 0 for 4MU-GL which very low as compared to logP of 9AC -GL and BQC -GL. This important factor needs to be considered to make generalization. Moreover, the application is only possible due to the lactone ring of the aminocamptothecin which is an important factor to consider for the switch based modification of molecule otherwise it is just another molecule as doxorubicin. This in turn makes the choice of 4MU improper. 6. Authors responded to the reviewer 2's comment on the dose selection of free and liposomal formulation. Though authors tried to justify the dose difference due to limitations of solubility, it would have been better to estimate the effectiveness at the same dose level as in clinical settings, iv anticancer drugs are always given by infusion which to some extent bypasses the solubility limitation. Anyhow, the dosing is also possible in the fractions, if authors had considered that. 7. Authors have given the in vivo activity of 9AC -GL liposomes against the free drug and parental drug (Fig 6). The results show that there is an unprecedented therapeutic activity of the liposomes which doesn't even allow the tumor to grow. Further, in Supplementary fig 10 a, the difference in tumor size between the Doxisome and 9AC -GL liposomes is unbelievable (~65 vs 0; i.e. >60 times). However, this doesn't coincide with the IC 50 assay in which IC 50 values are (2.4 vs 0.15, i.e. >24 times). Usually, the in vivo studies turn out to give results which most probably are lower as compared to in vitro and at the highest luck, same as in vitro results. The results need appropriate justification. 8. Release studies of the liposomes were performed in PBS. These studies need to be performed in three mediums (pH 7.4, pH 6.4 and pH 5.5) to represent the uptake of liposomes in the normal tissue, cancer interstitium and inside cancer cells. Reported release study is sufficient to justify the retention of the drugs in liposomes during circulation. 9. As authors also agree with the fact that EPR doesn't ensure total delivery of the nanocarrier to tumors, yet it is the only mechanism which is primarily important for preferential accumulation of nanoparticles in tumors as opposed to other normal tissues due to the leaky vasculature and inefficient lyphatic drainage in tumors. However, distribution to other normal organs is what cannot be avoided completely. Hence, given the very low IC 50s of the compounds, authors should consider a few in vitro studies to evaluate the cytotoxicity to normal cells i.e. liver cells, normal tissue cells (representative of the site of the cancer).

Response to reviewers
We wish to thank the reviewers for their careful examination of our revised manuscript. Their comments and suggestions have helped to improve the clarity and overall quality of our paper.
To help the reviewing process, we labeled in: -BLUE, our responses to the reviewers.
-RED, the modifications added to our manuscript.
-GREEN AND UNDERLINED, are the comments copied from previous reviewers.

Reviewer # 5
We thank reviewer #5 for helping us to improve our manuscript. Please find our responses to the expressed concerns below: 1. The addition of only 2 references mentioned by reviewer # 1 did not sufficiently addresses the problem regarding the novelty and needs to be expanded.
To clarify the novelty brought by the present work, we added the following text within the introduction: Additional information can be found at: Page 3, line 59: For example, docetaxel was modified by a N-methylpiperazinyl butanoic acid group to produce a protonated derivative to allow the use of a pH gradient for improved loading 20 . In another approach, docetaxel was modified by attachment of a glucose group to increase water solubility and improve loading capacity in liposomes by avoiding membrane accumulation of docetaxel 21 . Although glycosylated docetaxel retained 90.9 % of tubulin stabilization, such modification can strongly affect drug potency in other cases.

Page 4, line 73:
The method may be a more general approach as we demonstrated the utility of the switch concept by stably retaining chemically different hydrophobic drugs in liposomes.
2. Reviewer # 2 comments on the figures clarity and number were not sufficiently addressed Thank you for your comment. We tried to make the figures as clear and simple as possible, while retaining important data on the main figures without needing to frequently check the supplementary data. We modified the figures as follows: To help the reviewing, we wish to summarize here the changes that were made to the figure since our first version: Figure 1: We simplified the overall concept. The figure was split between a) and b) panels. Panel a) shows the general concept and panel b) shows in more details the case of 9AC-G W discussed within the paper. We removed unnecessary details.     Some additional changes that we made to simplify our figures are listed below: After modification, we believe that all of the current data displayed on the figures are necessary for the understanding of the fundamentals.
3. Reviewers # 1, # 2 and # 4 concerns were well addressed in the response letter but not in the manuscript, this needs addressing, especially for most of the fundamental and important questions and concerns contributed by reviewer # 4.
Thank you, we have listed below the changes we made to more completely respond to the reviewers comments.
Reviewer #1: If the drug solubility of 9-AC could be significantly improved to 30-40 mg/mL, why does the drug stock solution need to be prepared in DMSO at 10 mg/ml?" Drugs were dissolved in DMSO as stock solutions to prevent unwanted hydrolysis in water during storage. In addition, although the glycosidic switch drugs are soluble in water, the parental drugs are insoluble in water. We therefore dissolved all drugs in DMSO for a fair comparison and to remove the nature of the solvent as an experimental variable.
We modified the text to make this clear: Page 8, line 147: All drug stock solutions were prepared in DMSO to prevent undesired chemical hydrolysis during storage and to maintain a constant DMSO concentration of 9% (v:v) in the loading buffer for both water-insoluble parental drugs and water soluble glycosidic switch drugs. 3. DMSO has been shown to increase the membrane permeability, thereby enhancing the drug loading efficiency. Thus, the increased loading efficiency might be attributed to the presence of DMSO over the lipophilicity of the drug. This needs to be discussed." For this comment, we only discussed in the manuscript the fact that we used the same concentration of DMSO for all conditions. We now added more details about the suspected improved loading efficiency that could be attributed to the presence of DMSO.
Additional information can be found at: -Page 16, line 326: As DMSO is known to enhance the permeability of lipid bilayers, we used the same DMSO concentration (9%, v:v) for all the loading conditions to ensure that any improvement in drug loading efficiency is solely attributed to the influence of the glycosidic switch.
Reviewer #2: There is no direct evidence presented that the ester groups on the glucuronidated drugs are hydrolyzed inside the liposome. This must be shown. The release data in Figure 4D-F is suggestive, but not proof. Detailed new data to show this is needed." Data shown in Figure 3 and Supplementary Figure 2 provide direct evidence that the ester groups are hydrolyzed inside the liposomes. To make this clearer, we added the following sentence to the manuscript.
Additional information can be found at: -Page 15, line 309: As the liposomal fraction was separated from the external medium and lysed with detergent to reveal its contents, this provides direct evidence that the ester group of the lipophilic switch was hydrolyzed inside the liposomes.
"4. The data in Figure 6d is intriguing, but rather flawed. Clearly, the liposomal preparation wins out. However, it is compared with free drug, which is administered at 1/5th the dose due to solubility considerations. Given the vast improvement in exposure that the authors allude to, they should have included 2 mg/kg of the liposomal preparation for direct comparison." Actually, on a molar base of active drug, the difference between liposomal and soluble drug concentrations was 2.6 fold. The dose of liposomal drug was selected because we did not want to minimize the possible efficacy of 9AC-G liposomes due to the limitations of administrating free 9AC. The ability to administer more 9AC-G liposomes is an important advantage of this formulation. We included the reasons for this choice: Additional information can be found at: -Page 22, line 468: The chosen dosage was based on considerations of solubility and toxicity since we wished to test a condition representing a good compromise between therapeutic efficacy and toxicity.
"5. It is interesting that liposomal loading with the two camptothecins far exceeded that of 4MU. Why? What evidence do the authors have that the lactones in the camptothecin isn't playing a role, given that the vast majority would be in the hydrolyzed at pH 8.5? Furthermore, there is no discussion of even why 4MU is included in the paper at all and what was learned from the small amount of attention this particular compound seemed to have garnered." We included 4MU in our manuscript to demonstrate the benefice of the glycosidic switch for loading of compounds other than camptothecins. We believe that our strategy does not have to be restricted to a specific class of compounds like camptothecins, although the presence of the lactone ring might be beneficial. We believe that showing 4MU as an additional example provides an honest comparator as a model for non-camptothecin compounds. In addition 4MU might possess some interesting anticancer properties as mentioned in our manuscript.
We added some additional information at: Page 13, line 267: 4MU was chosen as a representative of "non-camptothecin" compounds to demonstrate the utility of the glycosidic switch in a more general approach.
More discussion was added to explain the lower loading efficacy of 4MU-G as compared to 9AC-G and BQC-G.
Page 16, line 328: 4MU-G L was loaded less efficiently than 9AC-G L and BQC-G L . We suspect that the partitioning of the modified drugs between the aqueous and the organic phases is important for efficient loading. For example, 4MU-G L is only very slightly lipophilic (Log P ~ -0.002) and it appears that a clear partitioning towards the organic phase is preferred, as observed with BQC-G L (Log P ~ 1.3) and 9AC-G L (Log P ~ 1.1) for efficient loading. We suspect that esterification of the glycosidic switch with longer carbon chains (i.e. ethanol, propanol, etc.) might help to increase the log P values of such compounds and therefore the loading efficiency. In the end, the loading efficiency of 4MU-G L was still superior to the parental compound 4MU (Fig. 3c).
Considering the effect of the lactone ring we also added the following: Page 17, line 363: A beneficial effect of the camptothecin lactone ring on improved retention can also be considered, as the lactone ring is present in the open charged carboxyl form at the high pH inside liposomes. However, the poor retention of 9AC and BQC in the same liposomes (with high internal pH) demonstrates that opening of the lactone ring by itself is insufficient to achieve good retention of the drugs inside liposomes.
"8. The Doxisome comparison in Supp. Figure 3  We agree that the EPR effect limits the amount of nanomedicine that can accumulate in tumors. For this reason, we suggest that that the potency of the loaded compounds plays a major role in the success of anticancer drug delivery in therapy. Doxorubicin is not very potent, which may be a major reason that Doxil is only effective for tumors with exceptional EPR. We therefore added the following to the discussion: Page 22, line 485: […] encapsulation of highly potent drugs in liposomes may be highly beneficial to help overcome inherent limitations in tumor drug accumulation afforded by the EPR effect. We believe that in order to counterbalance the low efficacy of EPR delivery, the potency of nanomedicines is a critical factor for successful tumor therapy. The recent demonstration of the benefits of nanoparticle drug delivery of a highly potent antiproliferative compound, monomethylauristatin E (MMAE), also supports this idea 1 . Since the potency of 9AC is about two orders of magnitude greater than doxorubicin, we suspect that the benefit obtained by 9AC-G W liposomes is significantly greater than with doxorubicin liposomes, as demonstrated by our in-vivo results.
"There are no clinical evidences available supporting that long-circulating stable nano-sized delivery systems outperform to short circulating unstable nano-formulations. Examples contrast Doxil vs. Myocet and NK105 vs. Genexol PM." A recent study published in Nature Communications showed that stable nano-formulations bring beneficial effects compared to unstable nano-formulations ("Augmenting drug-carrier compatibility improves tumor nanotherapy efficacy") 2 . In this work, nanoparticles with poorly retained drug had low drug to tumor accumulation (~ 0.1 % ID) and were not effective to treat cancer. On the other hand, nanoparticles with strongly retained drug could deliver drug more efficiently to tumors (> 1 % ID) and had significantly better antitumor activity and survival. We added the new reference to the manuscript: Page 3, line 55: This is important because stably retained drugs allow greater tumor accumulation than unstable drugs (> 1 % ID vs. ± 0.1 % ID) and consequently display stronger anticancer activity and overall survival in-vivo 2 .
"Technically, water-soluble drugs can be loaded in liposomes without active conversion from GL to Gw form during the hydration process." "The active loading may improve loading efficiency but compensated with chemistry involved. The reason for precipitation of Gw forms in the liposome is not clearly explained. Precipitation by the presence of divalent ions (Ca++) is not supported. It is, then, not clear the slow release kinetics is due to precipitation or water-solubility." It is very difficult to directly load water soluble versions of the drugs as shown by the low loading of the water soluble versions in Fig. 3c. We speculate that calcium ions help retain the drugs by precipitation, but the actual mechanism requires further study. However, regardless of calcium-mediated effects or increased water solubility, clearly the glycosidic switch greatly increases the retention of 9AC and BCG inside the liposomes as shown in Figure 4. We believe that drug stability is more important than the effect on loading. We added the following text as explanations: Page 17, line 345: The calcium ions in the liposomes likely form a complex with the glycosidic switch under a water soluble form (-G W ), by reaction between positively charged calcium and negatively charged carboxylate of the switch, leading to precipitation inside the liposomes, which may help retain molecular cargos during delivery 3 .
And more details were also provided about the potential effect of calcium: Page 17, line 363: A beneficial effect of the camptothecin lactone ring on improved retention can also be considered, as the lactone ring is present in the open charged carboxyl form at the high pH inside liposomes. However, the poor retention of 9AC and BQC in the same liposomes (with high internal pH) demonstrates that opening of the lactone ring by itself is insufficient to achieve good retention of the drugs inside liposomes.
"The in vivo study lacks a control of liposomal 9AC. The tumor is very slow growing even after inoculation of 10 million cells. The reason is not clear." We previously answered this concern by mentioning that we chose to compare 9AC-G W liposomes in-vivo with another stable long circulating liposome, and we selected doxorubicin liposomes for that purpose since they are one of the best characterized anti-cancer liposomal drug. As mentioned, 9AC liposomes did not show any stability in-vitro with near 90% of drug loss within just hours. That makes it behaves probably in a similar way as "free 9AC" which was included in our study.
Concerning doxorubicin liposomes, as demonstrated in supplementary figure 11, they were not able to reach a good therapeutic efficacy in mice without causing toxicity. Advantageously, this result contrasts with liposomal 9AC-G W .
We agree with the lack of details added to the manuscript concerning that point, and hope to make it clearer this time: Page 22, line 466: We chose doxorubicin liposomes as the closest control for 9AC-G W liposomes, as both are stable long-circulating liposomal formulations of anticancer compounds. The chosen dosage was based on considerations of solubility and toxicity since we wished to test a condition representing a good compromise between therapeutic efficacy and toxicity.
We also which to add some details about the slow growing rate of MDA-MB468 in mice xenograft models, we believe that such models are more representative of the real tumor growing rate in humans: Page 21, line 464: MDA-MB468 tumors grow slowly in mice, which might more closely mimic the growth rate of tumors in humans, and may therefore be a more appropriate model of cancer than faster growing xenografts.

Reviewer # 6
We thank reviewer #6. The comments were helpful to improve the manuscript. Please find our responses to the expressed concerns below: 1. Camptothecins have been noted to be very unstable in aqueous media due to their closed lactone ring, which opens at alkaline pH (pH>5.5) forming inactive open lactone form. The rates of hydrolysis of CPT in a PBS (pH 7.4) have been studied and it has been shown that only 10% activity remained after 2 hours in PBS. Moreover, liposome development has been targeted to stabilize the compounds in the bilayer where its lactone is protected from the hydrolytic aqueous phase. Keeping these in mind and use of pH 8. Thank you for this comment. It is true that 9AC-G W is present with an open lactone ring due to the high pH inside liposomes as drawn in figure 1. This is actually an advantage since the open lactone ring may further enhance the retention of drug inside liposomes beyond what is provided by the glycosidic switch. As mentioned by reviewer # 6, the opened lactone form of camptothecins display poor anticancer activity. However, in our case, the glycosidic switch is enzymatically removed after liposomes reach lysosomes in target cells (Fig 1, Fig 5 &  supplementary figure 7). The lactone ring in the drug can spontaneously reform due to the low pH in lysosomes (pH ~4.5). Thus, active drug is expected to be generated directly inside target cells, which may allow very effective inhibition of topoisomerase I.
We added the following summary to the revised manuscript: Page 14, line 289: It is worth noting that camptothecins drugs (such as 9AC and BQC) might be well suited for improved retention due to the presence of a lactone ring. Indeed, the lactone ring will be found under an open form at the high pH inside the liposomes, revealing an additional charged carboxylate group for enhanced retention.
2. Authors showed that the switch molecules are hydrolyzed to water soluble counterparts (Fig 2 Conversion of 9AC-GL to 9AC-GW), however,. they never emphasized on the fact that it is not actually the conversion of 9AC-GL to 9AC-GW and rather it is conversion to open lactone 9AC-GW. Authors need to bring this to notice in the manuscript and also explain the disposition occurring in the cancer cells under acidic conditions, which is essential for activity of 9AC (about lactone ring).
Thanks you for the suggestion to improve the clarity of our manuscript concerning the lactone-carboxy forms of the CPT drugs. In our manuscript, the term "9AC-G W " refers to both the closed or opened lactone forms. The open lactone is the form present inside liposomes. After being released in the lysosomes, the closed lactone form can spontaneously form due to the acidic pH conditions in lysosomes for escape into the cytosol and for improved potency.
Additional information was added to the manuscript: Page 8, line 159: For all analysis, camptothecin drugs were incubated at pH 2.9 before injection to allow the reformation of the lactone ring.
Page 13, line 258: We wish to emphasize that the term "-G W " used with camptothecin drugs refers either to the closed lactone or the opened carboxyl forms, which depends on the environmental pH. As an example, "9AC-G W " inside the liposomes, refers to the open carboxyl form, however, when released inside the lysosomes "9AC-G W " is expected to reform the closed lactone ring form.
Page 14, line 292: After lysosomal routing and enzymatic degradation of the liposomes 4 , the closed lactone form can spontaneously reform at the acidic pH in lysosomes for rapid escape into the cytosol and enhanced anticancer activity afforded by the lactone form of camptothecins 5 .
3. This in turn also warrants the explanation for further disposition and activity of other molecules (glucuronic acid, calcium and benzyl alcohol) generated at equimolar or higher concentrations as drug after the glucuronidase cleavage. Don't these molecules impart their effects on the cytotoxicity or other effects?
CPT drugs are orders of magnitude more potent than the other mentioned components of the switch drugs. To our knowledge, no toxicity of glucuronic acid has been observed or reported. One study specifically focused on the preclinical toxicological study of Dglucuronic acid 6 and concluded that: "A one-month treatment of rats (at a single daily dose of 50, 250, and 500 mg/kg, i.p.) and dogs (50 mg/kg, i.v.) induced neither functional nor morphological changes in hemopoietic and lymphoid organs, kidney, heart, as well as in the digestive, nervous, hemostatic, and fibrinolytic systems.". From their results, even very high doses of D-glucuronic acid only produced local irritation after intraperitoneal injection.
High calcium in blood results in a pathology called hypercalcaemia when levels are higher than 2.6 mmol per liters, a level which is expected to be higher than the dose received at therapeutic efficacy of the liposomes.
Benzyl alcohol was reported to have low acute toxicity with LD50 dose greater than 1 g per kg in animals. It also has been widely used as an antimicrobial preservative in medication at 0.9% for infants, but since 1982, the FDA issued recommendations to warn pediatricians against the use of benzyl alcohol containing fluids and diluents intended to be used in newborn infants whenever possible. This concern is due to the immature metabolic and excretory pathways in infants, particularly in low birth weight infants. However, at therapeutically effective dosage of drug-loaded liposomes, the amount of benzyl alcohol produced after beta-glucuronidase activation is estimated to be lower than its toxic dose, especially in adults. We made a simple estimation based on the therapeutic dosage (10 mg/kg): If 100% of 1% (targeting successfully the tumor) of the total liposomes injected were processed, it would produce only 2.9 nmol of benzyl alcohol which is equal to 15.6 µg/kg, thus almost 65000 times below the LD50.
Altogether we think that compared to the toxicity of the parental compounds (and especially camptothecins) the byproducts generated after enzymatic cleavage will only have a minor anti-proliferative effect.
We added the following to the manuscript: Page 20, line 430: Several byproducts of the degradation of the glycosidic switch, including benzyl alcohol and glucuronate, are produced during liposomal processing within the cells. However, their toxicity is several orders of magnitude below the toxicity of camptothecins.
4. Secondly, when authors have used methanol for esterification, this makes it important that authors explain the release of methanol upon hydrolysis. As methanol could be toxic to the cells, they need to rationally justify based on the quantity of the methanol that the cells will be exposed upon dosing and give relevant deduction.
Methanol is first used for the synthesis of the lipid soluble forms (G L forms) and is removed after HPLC purification by rotary evaporation. Traces of methanol are generated inside liposomes during internal conversion of the G L forms to G W forms at equimolar quantities. Methanol generated inside the liposomes can diffuse through lipid bilayers 7 and is diluted in the external buffer of the liposomes. Later, the methanol is removed in a similar way to the non-encapsulated drugs by gel filtration on a G50 size exclusion column. For this reason, the presence of methanol is unlikely when exposing the cells to loaded liposomes. If 100% of the methanol produced in the liposomes was retained, the dose of methanol that would be injected would be equal to 290 nmol for a 20 gr mouse, or only 9.3 µg (equal to 11.8 nL), at therapeutic effective dosage (10mg/kg).
We added the following to the manuscript: Page 15, line 315: After loading, the glycosidic switch is exposed to an internally high pH and undergo saponification, releasing methanol. As methanol diffuses through lipid bilayers 7 , it is not retained internally and is diluted in a larger external volume. Removal of nonencapsulated compounds after loading also contributes to remove the methanol generated during that step.
5. Authors' generalization to apply the switch methodology to a wide range of compounds is highly speculative. Even though 9AC, BQC and 4MU had similar log P values, their switch versions i.e. W and L versions gave different partitioning to the molecules with logP of 0 for 4MU-GL which very low as compared to logP of 9AC-GL and BQC-GL. This important factor needs to be considered to make generalization. Moreover, the application is only possible due to the lactone ring of the aminocamptothecin, which is an important factor to consider for the switch-based modification of molecule otherwise it is just another molecule as doxorubicin. This in turn makes the choice of 4MU improper.
It is likely that the presence of the lactone ring on camptothecin compounds helps for the retention of the drugs inside the liposomes. We observed as well that 4MU-G W could not be found at similar extent inside liposomes compared to 9AC-G W and BQC-G W . We have linked this result with the fact that the logP of 4MU-G L is not lipophilic enough to permit the drug loading to be as efficient as the other two compounds. We actually think that the use of 4MU-G provides honest estimation about the generalization of this method. In addition we also provided a comparison of the loading efficacy between parental 4MU and 4MU-G and observed a significant improvement of the loading capability using the glycosidic switch, also justifying its use for other compounds than camptothecins.
Additional information can be found at: Page 14, line 289: It is worth noting that camptothecin drugs (such as 9AC and BQC) might be well suited for improved retention due to the presence of a lactone ring. Indeed, the lactone ring will be found under an open form at the high pH inside the liposomes, revealing an additional charged carboxylate group for enhanced retention.
Page 17, line 363: A beneficial effect of the camptothecin lactone ring on improved retention can also be considered, as the lactone ring is present in the open charged carboxyl form at the high pH inside liposomes. However, the poor retention of 9AC and BQC in the same liposomes (with high internal pH) demonstrates that opening of the lactone ring by itself is insufficient to achieve good retention of the drugs inside liposomes.
Page 16, line 328: 4MU-G L was loaded less efficiently than 9AC-G L and BQC-G L . We suspect that the partitioning of the modified drugs between the aqueous and the organic phases is important for efficient loading. For example, 4MU-G L is only very slightly lipophilic (Log P ~ -0.002) and it appears that a clear partitioning towards the organic phase is preferred, as observed with BQC-G L (Log P ~ 1.3) and 9AC-G L (Log P ~ 1.1) for efficient loading. We suspect that esterification of the glycosidic switch with longer carbon chains (i.e. ethanol, propanol, etc.) might help to increase the log P values of such compounds and therefore the loading efficiency. In the end, the loading efficiency of 4MU-G L was still superior to the parental compound 4MU (Fig. 3c). 6. Authors responded to the reviewer 2's comment on the dose selection of free and liposomal formulation. Though authors tried to justify the dose difference due to limitations of solubility, it would have been better to estimate the effectiveness at the same dose level as in clinical settings, iv anticancer drugs are always given by infusion which to some extent bypasses the solubility limitation. Anyhow, the dosing is also possible in the fractions, if authors had considered that.
We are considering deeper research about in-vivo activity of glycosidic switch related drug loading. For this article, we have focused more about the proof of concept and we understand that further animal studies are necessary for future directions. As mentioned previously, we chose dosage based on the maximum tolerance dose, in order to show the maximum benefit that we could acquire from each group tested We added the following to the manuscript: Page 22, line 468: The chosen dosage was based on considerations of solubility and toxicity since we wished to test a condition representing a good compromise between therapeutic efficacy and toxicity.
7. Authors have given the in vivo activity of 9AC-GL liposomes against the free drug and parental drug (Fig 6). The results show that there is an unprecedented therapeutic activity of the liposomes, which doesn't even allow the tumor to grow. Further, in Supplementary fig 10 a,  We noticed this phenomenon as well, and we believe that this is due to the potency of the drug loaded inside the liposomes. For example, in the case of doxorubicin, the potency is relatively low, and it resulted in no additional clinical benefit compared to free drug, excepted from cardioprotection effect. The enhanced permeability and retention effect, which is so far the only mechanism to target nanoparticles passively to tumors, is relatively poor in terms of efficiency (around 1% of total injected dose target tumors at most). Consequently, doxorubicin liposomes in-vivo would not be able to display as good efficiency as liposomes loaded with a more potent drug, such as camptothecins. For the same amount of targeted liposomes, doxorubicin might not be able to inhibit tumor cells, as they would do in an invitro assay where all the nanoparticles are exposed directly to the cells. On the other hand, even with low targeting efficiency, liposomes loaded with camptothecins would potentially be able to inhibit tumor growth in a much more effective way. This phenomenon was discussed and published recently using monomethyl auristatin conjugated nanoparticles.
Additional information can be found at: Page 22, line 481: The in-vivo anticancer activity of 9AC-G W liposomes was surprisingly more effective than Doxisome; 9AC-G W liposomes cured tumors even though the difference in the in-vitro IC 50 values of 9AC-G W liposomes and Doxisome was only about 20 fold (Supplementary table 1). This result suggests that encapsulation of highly potent drugs in liposomes may be highly beneficial to help overcome inherent limitations in tumor drug accumulation afforded by the EPR effect. We believe that in order to counterbalance the low efficacy of EPR delivery, the potency of nanomedicines is a critical factor for successful tumor therapy. The recent demonstration of the benefits of nanoparticle drug delivery of a highly potent antiproliferative compound, monomethylauristatin E (MMAE), also supports this idea 1 . Since the potency of 9AC is about two orders of magnitude greater than doxorubicin, we suspect that the benefit obtained by 9AC-G W liposomes is significantly greater than with doxorubicin liposomes, as demonstrated by our in-vivo results. In a similar way, early antibody-drug conjugates (ACDs) that employed unstable linkers to attach moderately potent anticancer agents such as doxorubicin or methotrexate produced disappointing clinical benefits 8,9 , whereas later ADCs that employed stable linkers to attach highly potent drugs have displayed spectacular clinical anticancer activity 10,11 .
8. Release studies of the liposomes were performed in PBS. These studies need to be performed in three mediums (pH 7.4, pH 6.4 and pH 5.5) to represent the uptake of liposomes in the normal tissue, cancer interstitium and inside cancer cells. Reported release study is sufficient to justify the retention of the drugs in liposomes during circulation.
We understand the concern. However, we worry about misleading our audience if performing a release assay at a lower pH. Our concern is that the readers might get the wrong impression that we are trying to claim that the liposomes are made so that they can release their cargo at low pH environment (i.e. lysosomes). In reality these liposomes are very likely to be stable at lower pH. We believe that the mechanism of cargo release inside the cells is due to the enzymatic machinery located inside the lysosomes (i.e. lipases) rather than the low pH itself.
We added the following to the manuscript: Page 14, line 292: After lysosomal routing and enzymatic degradation of the liposomes 4 , the closed lactone form can spontaneously reform at the acidic pH in lysosomes for rapid escape into the cytosol and enhanced anticancer activity afforded by the lactone form of camptothecins 5 .
9. As authors also agree with the fact that EPR doesn't ensure total delivery of the nanocarrier to tumors, yet it is the only mechanism, which is primarily important for preferential accumulation of nanoparticles in tumors as opposed to other normal tissues due to the leaky vasculature and inefficient lymphatic drainage in tumors. However, distribution to other normal organs is what cannot be avoided completely. Hence, given the very low IC50s of the compounds, authors should consider a few in vitro studies to evaluate the cytotoxicity to normal cells i.e. liver cells, normal tissue cells (representative of the site of the cancer).
Thank you for raising this important question. We measured the in-vitro anti-proliferation activity of 9AC-G W loaded liposomes with non-cancerous human fibroblasts (GM637), BALB/c normal liver cells (BNL CL.2) and mouse fibroblasts (3T3). We noted that the IC 50 value toward human fibroblasts was higher than any of the cancerous cells tested (1.6 µM vs. 0.1 ~ 1.2 µM) while murine normal fibroblast and liver cells IC 50 values were even greater, 16 and 22 µM, respectively. We believe that 9AC-G W liposomes demonstrate less toxicity to normal cells due to 2 main reasons: 1. The doubling time of normal cells is usually slower than cancerous cells, and 2. Cancerous cells overexpress lysosomal enzymes such as betaglucuronidase.
Additional information can be found at: Page 20, line 422: We also examined the anti-proliferative effect of 9AC-G W liposomes to "normal" human fibroblasts (GM637), murine liver cells (BNL CL.2) and murine fibroblasts (3T3) (Supplementary fig. 9). We noted that the IC 50 values of human fibroblasts treated with 9AC-G W liposomes was higher than any of the human cancer cells tested (1.6 µM vs. 0.1 ~ 1.2 µM) while murine fibroblasts and liver cells were even less sensitive to 9AC-G W liposomes with IC 50 values of 16 and 22 µM, respectively. We believe that 9AC-G W liposomes demonstrate reduced toxicity to normal cells due to two main reasons: 1) The doubling time of normal cells is usually slower than cancerous cells, and 2) The overexpression of lysosomal enzymes such as beta-glucuronidase by cancerous cells 12 .