Peer review of: Light-triggered switching of liposome surface charge directs delivery of membrane (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The manuscript entitled "Light-triggered switching of liposome surface chargedirects 
theintracellular delivery of membrane impermeable payloads in vivo" by G. Arias-Alpizara et al. 
describes light-induced switching of surface charges of liposomal carriers to induce cellular uptake 
of long-circulating membrane vesicles. Proof-of-concept is provided using a zebrafish embryo in 
vivo model. This novel concept is very interesting in that surface charge switching using photo-
activated lipids can be used to change the pharmacokintic properties of circulating nanoparticles 
within the living organism. 
 
Major comments: 
line 194: Content leakage of liposomes was monitored over a time period of 30 minutes (Figure 
S6). What about stability over prolonged periods of time (e.g. storage at low temperature) of non-
irradiated samples or irradiated samples during 24 hours? 
 
line 217: "Having previously shown photoswitching of liposome surface charge occurred within 
seconds": please provide the reference 
 
Liposome aggregation in vivo due to changes in the protein corona before or after irradiation will 
trigger cellular uptake by macrophages. Were DLS experiments carried out in presence as well as 
absence of plasma proteins? 
 
Please explain the advantages and limitations of the used zebrafish model in more detail. Is it 
possible to extrapolate from data obtained in zebrafish to higher vertebrates? 
 
Minor comments: 
Line 85: "In our experience, anionic nanoparticles...": please provide the reference or does this 
relate to ref. 25? 
line 98: "In the contrasting fates..." should read "The contrasting fates..." 
line 106: "emerging technologies to facilitate the administration of light deep within patients": 
please provide some background information on state of the art technologies (light guided devices, 
penetration depth of light within tissues as a function of wavelength, accessibility of tissues such 
as skin or inner organs) 
Line 121: Sedction title is "Results and Discussion". Line 314: This section is entitled "Discussion". 
Should this rather be "Conclusion"? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript reports on liposomes that modify their surface charge from neutral to cationic 
when they are exposed to light (365 nm) as an external, controllable stimulus. The authors have 
demonstrated both in vitro and in vivo (zebra fish) the feasibility and relevance of such a change 
in surface charge regarding liposomes circulation in blood and uptake by either endothelial cells or 
blood resident macrophages by adjusting the applied light dose. The introduction provides a clear 
picture of the state of the art and the relevance of the present study. The rationally behind the 
work is also clearly explained in the results and discussion section. The movies are also helpful for 
the understanding of the results. The experiments are described in detail. 
 
Can the authors provide the pKa of the amino moieties generated after light irradiation? What is 
the expected percentage of positively charged moieties compared to the total of amino groups in 
each liposome? According to Figure 1, the amino group is always positively charged, which maybe 
not accurate at pH 7.4. 

 
Lines 332-335. Our data indicates that above a certain cationic surface charge threshold, i.v. 
administered nanoparticles will predominantly “stick” to (and be internalised by) endothelial cells, 
particularly in blood vessels with reduced blood flow velocity, while below this charge threshold, 
nanoparticles will tend to aggregate in circulation and be subsequently cleared by the RES. Please 
define the threshold value and provide a hypothesis for aggregation. 
 
The comment on the replacing 365 nm light with NIR light to irradiate profound organs lacks of 
analysis of feasibility. Please comment on the challenges of finding groups that can be readily 
broken under NIR light. Please check what value of wavelength is the correct (365 nm or 375 nm 
as stated in the supporting info). 
 
Figure 6, plot g. The quality should be improved. 
 
Do the authors record any increase in temperature during exposition of the liposomes to the 375 
nm light? 
 
 
 
Reviewer #3: 
Remarks to the Author: 
General 
 
In the manuscript by Arias-Alpizar et al., the authors report on the development of liposomes of 
which the surface charge can be switched from neutral to cationic upon irradiation with UV light. 
The authors evaluate the photoactive liposomes in zebrafish models to determine the effects of 
surface charge switching on liposomal circulation time and uptake in endothelial cells and 
macrophages. 
The paper is well written with appropriate literature references and results presented in a clear 
manner. The study’s main feature is the demonstration of light-triggered switching of the liposome 
surface charge in vivo and imaging the effects in real time. While this proof-of-concept is of 
interest, the translational value and applicability of the approach can be subject to debate. This 
reviewer appreciates that the authors advocate for a thorough understanding of nano-bio 
interactions in vivo and argue that their approach shows that effective nanoparticles do not 
necessarily require complex designs. However, the ability to govern the biodistribution and cellular 
interactions of therapeutic nanoparticles (with straightforward composition) based on surface 
charge has been demonstrated by others, especially in the field of nucleic acid therapeutics (e.g. 
by Kranz et al. Nature 534, 396–401, 2016). In my opinion, the manuscript could be suitable for 
publication, provided several comments are addressed. 
 
Specific 
 
Specific 
 
1. The authors mention in the Introduction section that based on their previous studies (reference 
25) and experience, “anionic nanoparticles (<-20 mV measured zeta potential) interact strongly 
with RES cell types, namely scavenging endothelial cells (SECs, via a stabilin-mediated clearance 
pathway) and blood resident macrophages”. Have the authors also designed photocaged anionic 
lipids and determined liposomal cell uptake in zebrafish following irradiation? 
 
2. The authors use fluorescently-labeled lipids or sulforhodamine B (SRB) as a fluorescent model 
payload to determine liposomal localization and cell uptake. These approaches provide qualitative 
rather than quantitative results and do not provide information if the delivered payload is 
functional. Would the authors be able to show functional payload delivery, for example using a 
reporter model such as Cre-Lox? 

 
3. The authors mention in the final sentence of the Discussion section that “it is our hope that this 
study, and in particular the tools and methods employed, will expedite a transition from the 
empirical design of increasingly complex nanomedicines to the rational design of new, simple and 
effective nanomedicines”. How predictive/translational are the approach and the findings? It would 
strengthen the manuscript if this could be demonstrated. Would it for example be possible to 
irradiate (fluorescent) liposomal formulations prior to systemic administration in mice and 
determine cell uptake in macrophages and endothelial cells using flow cytometry compared to non-
irradiated formulations?