Peer review of: Use of ratiometrically designed nanocarrier targeting CDK4/6 and autophagy pathways for effecti (2020)

Reviewers' comments:  
Reviewer #1 (Expertise: NPs, drug delivery, Remarks to the Author):  


In this manuscript, the authors used a liposomal mesoporous silica nanocarrier for co-loading of a 
CDK4/6 inhibitor (PAL) and an autophagy inhibitor (HCQ) for pancreatic cancer therapy. There are 
several highlights in this manuscript as following:  

1. PAL and HCQ are highly synergistic in inhibiting pancreatic cancer cell proliferation at certain 
ratios. By utilizing an optimized remote loading method, PAL and HCQ can be ratiometrically loaded 
into the liposomal mesoporous silica nanocarriers.  

2. In vivo pharmacokinetics and intratumoral drug concentration tests confirmed that these two 
drugs were kept at designed ratios in the blood and the tumor tissue.  

3. The nanocarrier-enabled ratiometric drug delivery resulted in superior tumor inhibition effect 
over other forms in murine pancreatic cancer models.  

The authors show strong evidence that the ratiometrically designed nanocarrier will greatly enhance 
the therapeutic effect of PAL and HCQ over free combinations. This is quite meaningful since the first 
ratiometric liposome (Vyxeos®) for co-delivery of daunorubicin and cytarabine has been approved by 
the FDA in 2017.  

The manuscript is well organized and well written. In each part, the descriptions are extensively 
supported by detailed data.  

One concern for this manuscript is the payload chosen in utilizing this nanocarrier for cancer 
therapy. Besides biological aspects, chemical properties may be another reason for co-loading PAL 
and HCQ into the nanocarrier with the described remote-loading method. It will be more meaningful 
to give a comprehensive description on the ratiometric drug loading capacity of this nanocarrier in 
other drugs if more drugs are tested in the drug loading experiments.  



Reviewer #2 (Expertise: PDAC, autophagy, therapy, Remarks to the Author):  
Marginal success with CDK4/6 inhibitors in pre-clinical studies has prompted to develop combination 
therapies with better drug delivery methods to treat solid tumors. In this manuscript, Ji et al have 
used combination of CDK4/6 inhibitor Palbociclib (PAL)with autophagy inhibitor Hydroxychloroquine 
(HCQ) in a ratiometrically designed mesoporous silica nanoformulation(nanoparticle or NP) and 
showed that this method of drug delivery is more potent in part due to improved stability and bio-
availability of the drugs in tumors.  

Although this nanoparticle-based formulation shows better efficacy as compared to free PAL and/or 
HCQ, they observed tumor recurrence because of the expression of BCL-xl anti-apoptotic machinery. 
Combining a pan BCL2 inhibitor (ABT-737) with NP(PAL+HCQ); the authors could improve the 
antitumor effect of NP and suggested a rationale to use this three-drug combination for pancreatic 
cancer therapy. While this article is important and provides evidence of an efficacious drug delivery 
method, the concept of using PAL+HCQ is has been shown in other solid tumor studies ( for example 
Nature Communications volume 8, Article number: 15916 (2017)). Use of limited number of cell lines 
and missing controls in their experiments are major concerns of the paper. Immune cells have 
profound roles in the growth of KRAS driven pancreatic cancer (PDAC). As CDK4/6i(+MEKi) mediated 
growth inhibition of KRAS mutant tumors involves of immune cells, (Science. 2018 Dec 
21;362(6421):1416-1422. doi: 10.1126/science.aas9090), the formulation should be tested in 
immune-competent animal models. Following suggestions/ concerns need to be addressed to 
improve the manuscript and novelty of the study.  

1) The majority of the experiments have been done in only one cell line (PANC-1). As PDAC cells are 
very heterogeneous, the in-vitro study should be conducted in at least 5-6 cancer cell lines.  

2) The experiments should be performed in orthogonal methods to substantiate the conclusions. 
Immunoblots should be provided for Fig 1B, 2J, 5B and 5D. Please show p62 in immunoblot as well.  

3) Fig 4C. : As presented; the NPs accumulate in near CD31+ tumor blood vessels. How does the drug 
reach in the CD31 negative areas? Do those areas maintain proliferation? A co-staining with Ki67 
would be more informative.  

4) The histology sections are presented only in higher magnification. As this can be subjective, the 
authors should provide lower magnification views for a better assessment. A proper quantification is 
missing and should accompany every histological analysis.  

5) Many studies have shown CDK4/6 inhibition alone or combined with other drugs invoke a 
senescence response. Senescence is associated with the upregulation of BCL2 family of proteins. In 
this study, the authors found BCL-xl is upregulated; however, senescence is not associated with their 
drug treatment. This is an important observation that needs to be substantiated with proper 
controls. A) In Fig S10, a proper positive control for SA-B-gal staining should be presented to show 
that the assay is working in their setting. B) As discussed before this phenomenon should be proved 
in multiple cell lines. C) The authors can perform quantitative RT-PCR to show that senescence-
associated secretory phenotype (SASP) related transcripts are not regulated in their assays.  

6) As discussed, PDAC has a unique fibrous micro-environment. Not only it is impermeable to drug 
formulations, but also the cross-talk of immune cell and tumor cell provide a challenge to treat 
PDAC. As authors claim a more potent drug delivery method, the efficacy of NP or NP+ABT737 
should be performed in immunocompetent PDAC models (i.e. Kras-p53 GEMMs) or syngeneic 
orthotopic transplantation models.