European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Potential drug delivery nanosystems for improving tumor penetration T

⁎
Feifei Peng, Ruirui Li, Fang Zhang, Li Qin, Guixia Ling, Peng Zhang
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China

A R T I C LE I N FO A B S T R A C T

Keywords: Nanosystems, as one of the most important drug delivery systems, play a crucial rule in tumor therapy. However,
Potential the deep tumor penetration is retarded by the tumor physiological factors and nanomedicine properties. In this
Delivery review, we firstly elaborate the factors which impact tumor penetration, including the tumor physiological
Nanosystems factors and nanomedicine properties. Then, the latest and potential drug delivery nanosystems for improving
Penetration
tumor penetration are summarized and analyzed in detail. Moreover, recent combination therapies for im-
Therapies
proving penetration are described to enhance penetration. Finally, we summarize the typical clinical therapies of
potential drug delivery nanosystems.

1. Introduction past decades. There are a variety of treatments for anti-cancer, in-
cluding chemotherapy [1], immunotherapy [2], radiotherapy [3] and
Cancer has been the deadliest disease in many countries over the surgical resection [4]. In terms of chemotherapy, chemical drugs have

Abbreviations: AuNC@CBSA, small-sized renal-clearable red emission cationic bovine serum albumin-protected gold nanocluster; AuNCs, gold nanoclusters; AuNPs,
gold nanoparticles; AuNRs, gold nanorods; Bcl-2, a kind of antiapoptotic genes; Br-MSN, bromelain-conjugated mesoporous silica nanoparticle; BSA, bovine serum
albumin; CAF, cancer-associated fibroblast; CCM, cancer cell membrane; Ce6, chlorine e6; CD13, the receptor of iNGR which improves the capacity of tumor vessel
targeting; CD44, cluster determinant 44; CED, convection-enhanced delivery; CT, computed tomography; CUR, curcumin; DACHPt, 1,2-diaminocyclohexane-pla-
tinum(II); DC101, antivascular endothelial growth factor receptor-2 antibody; DGL, dendrigraft poly-lysine; DMA, dimethylmaleic anhydride; DOX, doxorubicin;
DpNG, biodegradable cationic paclitaxel-loaded nanogel; DSPE, 1,2-distearoyl-sn-glycero −3-phosphoethanolamine; DTX, docetaxel; DTX-pHPM, DTX-pH polymeric
micelle; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EPR, enhanced permeability and retention; ESMSV, enzyme-stimulated multistage
vector; FAP, fibroblast activation protein; FDA, Food and Drug Administration; FITC, fluorescein isothiocyanate; GBM, glioblastoma multiforme; GEM, gemcitabine;
GNPs, gelatin nanoparticles; GSH, glutathione; GSSG, glutathione disulfide; HA, Hyaluronan; HAase, hyaluronidase; HCC, hepatocellular carcinoma; HIFU, high
intensity focused ultrasound; HMSNs, hollow mesoporous silica nanoparticles; HN, NIR laser sensitive nitric oxide donor; HSA, human serum albumin; HSC, he-
matopoietic stem cell; HT, hyperthermia; ICG, indocyanine green; IDD, small-sized dendrimer prodrug; IFP, interstitial fluid pressure; IL-24, interleukin-24; ILP,
isolated limb perfusion; iNGR, a type of tumor penetrating peptides, which harbors NGR and CendR motifs; iNGR-SSL, iNGR-modified sterically stabilized liposome;
IP, intraperitoneal; iRGD, a type of tumor penetrating cyclic peptides, which harbors the RGD αv-integrin-binding motif and the RGDK cryptic CendR motif; iRNP,
iRGD nanoparticle; KLAK, cytotoxic peptide; Lac, lactose; LCP-QP NP, lipid/calcium /phosphate nanoparticle; MC, merocyanine; MCSs, multicellular spheroids;
MMPs, matrix metalloproteases; MNC, micelle nanocluster; MPEG, methoxy poly (ethylene glycol); MRI, magnetic resonance imaging; Ms-DDS, multistage drug
delivery systems; m-SR-XRF, m-synchrotron radiation X-ray fluorescence; MTX, methotrexate; NDDs, nano-drug delivery system; NIR, near infrared; NLSC, N-lysinal-
N, -succinyl chitosan; NP, nanoparticle; nRGD, one of the iRGD derivatives; NRP-1, neuropilin-1; ODN, oligodeoxynucleotides; AP, acetylated pullulan; PA, pho-
toacoustic; PAE, poly (β-amino ester); PAEMA, poly (2-azepane ethyl methacrylate; PCL-b-PVP, poly (epsilon-caprolactone)-b-poly (N-vinylpyrrolid one); PCL-CDM-
PAMAM, poly (amidoamine)-graft-polycap rolactone; PDGF, platelet-derived growth factor; pDNA, plasmid DNA; PDNP, prodrug nanoparticle; PDT, photodynamic
therapy; PEG, polyethylene glycol; PEI, polyethyleneimine; PLA, poly lactide; PLGA, poly (D,L-lactide-co-glycolide); PLGLAG, MMP2-sensitive peptide; PNIPAM, poly
(N-isopro pylacrylamide); PPMEMA, poly 2-(penta methyleneimino) ethylmethacrylate; PS, photosensitizer; Pt, platinum; PTT, photothermal therapy; PTX, pacli-
taxel; QDs, quantum dots; RBC, red blood cell; RES, reticular endothelial system; rHuPH20, recombinant human hyaluronidase PH20; ROS, reactive oxygen species;
RRGD, a tandem peptide of RGD; Sf, sorafenib; siRNA, small interfering RNA; SNP, shell-stacked nanoparticle; SP, spiropyran; TAM, tumor-associated macrophage;
TAT, cell penetrating peptide; TGF-β, transforming growth factor beta; TGF-β-I, transforming growth factor-β inhibitor; THNC, “Trojan Horse” nanocarriers; TME,
tumor microenvironment; TNF-α, tumor necrosis factor-α; TPGS, tocopheryl polyethylene glycol succinate; TSL, thermosensitive liposome; UCL, up-conversion
luminescence; UCNPs, up-conversion luminescent nanoparticles; UCPZ, up-conversion nanoparticles-platinum-ZnFe2O4; VEGF, vascular endothelial growth factor;
Wnt16, a member of the wingless type MMTV integration site family
⁎
Corresponding author.
E-mail addresses: 1134592416@qq.com (F. Peng), 836840641@qq.com (R. Li), 745707048@qq.com (F. Zhang), 1639625350@qq.com (L. Qin),
ajps2006@163.com (G. Ling), zhangpengspu@163.com (P. Zhang).

https://doi.org/10.1016/j.ejpb.2020.04.009
Received 14 June 2019; Received in revised form 2 March 2020; Accepted 11 April 2020
Available online 18 April 2020
0939-6411/ © 2020 Elsevier B.V. All rights reserved.
F. Peng, et al.
been widely used to prevent the proliferation, infiltration, and metas-
tasis of cancer cells until eventually killing the cancer cells. However,
the low selectivity and poor water solubility of chemical drugs restrict
their further clinical applications [5]. Recently, a myriad of nanosys-
tems have attracted extensive attention for cancer treatment, which are
achieved by the passive targeting (enhanced permeability and retention
(EPR) effect) and/or active targeting (surface modification of na-
noagents with targeting moieties) principles. There exist three phases in
drug-loaded nanoparticles delivery: blood circulation, tumor penetra-
tion, and interaction with the targeted tumor cells. To maximize the
therapeutical efficiency, nanoparticles (NPs) must spread throughout
the entire tumor from the peripheral to the central regions. Un-
fortunately, solid tumors are characterized by abnormal tumor vascu-
lature, high density of cells and extracellular matrix (ECM), the ele-
vated tumor interstitial fluid pressure (IFP) and so on [6]. The
components of tumor microenvironment together form a big biological
barrier for nanoparticle deep penetration and subsequently weaken the
potency of drugs used in the tumor parenchyma.
On the other hand, the penetration efficiency of nanodrugs is sig-
nificantly affected by the physicochemical properties of nanoparticles
(size, shape, surface charge as well as ligand conjugation). The nano-
particle intratumoral transport depend on the slow diffusion, so its size
is identified as a critical design parameter that impacts their distribu-
tion [7]. It was believed that smaller nanoparticles were delivered to
the core areas of the tumor mass and showed higher performance on the
aspect of intratumoral penetration ability [8]. But too small particles
(their diameters are less than 5 nm) were cleared from the renal, thus
shortening blood circulation time and compromising tumor targeting
efficiency [9]. Therefore, the choice of size is involved with seeking
equilibrium between the extended blood circulation time in the normal
tissues and the enhanced penetration in tumor tissues. To overcome the
above difficulties and improve the delivery efficiency, multistage drug
delivery systems (Ms-DDSs) which are endowed with switchable size
have been exploited by many researchers to enhance tumor penetra-
tion. In other words, these nanoassemblies have appropriate char-
acteristics that ensure the prolonged circulation and accumulation in
blood vessels, and convert to smaller particles for better penetration
capability in tumors. The stimuli utilized to trigger the size transfor-
mation of nanoagents can be endogenous factors or external factors.
In this review, we firstly elaborate tumor physiological factors and
nanomedicine properties which impact tumor penetration. Then, the
recent strategies to fight against cancers through overcoming tumor
penetration barriers are described in detail. Finally, the combination
therapies and typical clinical therapies are also discussed.
2. Tumor physiological factors that impact tumor penetration
Compared with normal tissues, tumors usually have unique struc-
tural characteristics. Besides, different cancer tissues vary in biological
characteristics, vasculature and stroma. Some can be reached well by
nanomedicines (such as Kaposi sarcoma), others are challenging (such
as pancreatic cancer [10]). The latter particularly require strategies to
increase penetration. Factors which hinder the in-depth transport of
nanomedicines include abnormal tumor vasculature; elevated IFP;
dense ECM; cancer-associated fibroblasts (CAFs); tumor-associated
macrophages (TAMs) and hypoxia (Fig. 1).
2.1. Abnormal tumor vasculature
In the development of healthy tissues and cells, the formation of
blood vessels is carefully regulated to ensure that all cells can get ample
oxygen and nutrients by molecular diffusion [11]. Malignant tumors are
formed irregularly. They must acquire their own blood supply to
achieve rapid proliferation through angiogenesis. Therefore, the struc-
ture of tumor vasculature is poorly-organized, directly leading to the
more penetration capacity of tumor tissues than healthy ones [12]. To
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
normalize tumor blood vessels, the application of antiangiogenic drugs
is necessary to resist the abnormal angiogenesis.
In contrast to the healthy vessels, there are too many leakages in
tumor vessels, which are caused by large pores in blood vessels. In the
system circulation, nanoscale formulations tend to accumulate in the
leaked areas of the tumor. It is commonly described as “EPR effect”
which is magnified by the increased retention of tumor lymphatic
drainage [13]. It is worth mentioning that, it guides nanotherapeutics
to enter tumor vasculature more rapidly than other organs including
the liver, spleen, and lungs. Although the abnormal tumor vasculature
confers an advantage for the accumulation of NPs, the transshipment
ability of the emerging nanomaterials still poses a significant challenge
to diffuse into the surrounding tumor tissues and deep areas.
2.2. Elevated interstitial fluid pressure (IFP)
Besides, factors that hinder the penetration of nanodrugs include the
elevated IFP. The negative IFP, which is required to promote fluids flow
and transport nutrients from blood vessels into the cells of normal tis-
sues [14]. In terms of some solid tumors, however, there is a sig-
nificantly elevated IFP which is resulted from the abnormal perme-
ability, along with high cell density and the insufficient lymphatic
drainage in the tumor interstitial space [15]. As a result, the transport
of therapeutic agents is largely discouraged by the unfavorable pressure
gradient between the tissues and blood vessels. Even worse, elevated
IFP may cause substances in the tumor to flow back into the blood
capillaries, including NPs, cancer cells and growth factors, thus accel-
erating tumor growth and metastasis [16]. Recently, antagonists of
platelet-derived growth factor (PDGF), transforming growth factor beta
(TGF-β), vascular endothelial growth factor (VEGF) and a multikinase
inhibitor have been used to decrease IFP and improve the intratumor
transport of nanomedicines.
2.3. Dense extracellular matrix (ECM)
ECM is mainly comprised of a crosslinked net of collagen, elastin
fiber, proteoglycan and hyaluronic acid (HA) [17]. It is one of the
biological barriers to penetrate malignant cells. In tumors, the collagen
content is obviously higher than that in healthy tissues, which forms
relatively compact extracellular spaces. The dense collagen network
renders nanodrugs immediately localize in the surrounding vascular
areas and severely impairs their mobility [18]. In addition, the charged
substrates, such as collagen and HA, impede the delivery of positively
charged nanomedicines owing to electrostatic interactions [19–20]. By
degrading or normalizing ECM, some researchers have successfully
improved the penetration of nanomedicines.
2.4. Other factors
The tumor microenvironment (TME) is also characterized by the
overexpression of cancer-associated fibroblasts (CAFs), tumor-asso-
ciated macrophages (TAMs) and hypoxia. CAFs and TAMs, the major
cell types in tumor stroma, prevent the nanomedicine delivery and lead
to restricted response of solid tumors. Hypoxia, a prominent feature of
TME, is ascribed to rapid cell proliferation and abnormality in tumor
blood vessel structure. In addition, inadequate oxygen supply occurs in
the center of the tumor away from the microvessels, hindering the NP
penetration into deep tumor regions [21].
3. Nanomedicine properties that impact tumor penetration
3.1. Size
Particle size of nanodrugs is one of the most critical features which
govern their vivo fate, including blood circulation kinetics, tumor ac-
cumulation and deep penetration. NPs whose size are smaller than
F. Peng, et al.
Fig. 1. Overview of tumor unique structural characteristics.
10 nm are easily excreted by the kidneys, whereas larger particles are
usually captured by reticular endothelial system (RES) during circula-
tion [22]. In a pioneering research conducted by Kataoka and cow-
orkers, 18 nm small interfering RNA (siRNA) complexes, which abro-
gated the need for larger NPs, were engineered for the delivery to
stroma-rich fibrotic pancreatic cancers and orthotopic brain tumors.
The particles with appreciably small size comparable to antibodies,
combined stable systemic circulation with enhanced tumor penetration
[23]. Furthermore, the similar connection between particle size and
tumor penetration had also been validated in a series of NPs e.g.
polymeric NPs [24], mesoporous organosilica NPs [25], ultrasmall gold
NPs (AuNPs) [26], and PAMAM dendrimers [27]. All results jointly
indicate that smaller NPs have more potential in deep tumor penetra-
tion.
3.2. Surface charge
Besides particle size, surface charge is also an influential element for
improved tumor penetration and tumor therapy. Because the cationic
NPs have the tendency to interact with negative-charged matrix poly-
mers such as collagen and proteoglycans, cationic materials penetrate
more deeply than the anionic counterparts [28]. It was demonstrated
that positively charged poly (D, L-lactide-co-glycolide) (PLGA) NPs
achieved better tumor penetration than the negative ones (-13 mV) in
the xenografts [29]. However, the cationic ones were prone to be op-
sonized and cleared by RES during blood circulation. Compared with
the positive or neutral NPs, the negative ones were more effective on
the aspect of penetration in the liposomes [30] and gold nanorods
(AuNRs) [31]. In short, the relationship between surface charge and
nanomedicine penetration is too complex to elaborate fully.
3.3. Shape
Particle shape is also a crucial parameter that influence blood cir-
culation and tumor penetration. More and more attention has been paid
to nonspherical vehicles, which penetrate deeper than spherical mate-
rials [32]. In the latest study, the cancer cell membrane (CCM)-coated
NRs displayed more rapid extracellular matrix penetration than sphe-
rical particles and shipped doxorubicin (DOX) to the nucleus [33]. The
unique yolk-shell CCM-camouflaged NPs frequently transformed into
ellipsoidal shape particles, facilitating the penetration throughout
multicellular spheroids and yielding 95% tumor-inhibition rate [34].
Taken together, rational shape design is essential to improve antitumor
therapy.
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
3.4. Targeting moieties
The actively-targeted nanovehicles are a landmark discovery in
cancer therapy. After ligand-modified NPs go through several barriers
which are generated by TME, cell internalization and therapeutical
efficiency are consequently enhanced by ligand-receptor binding. It
seems that high ligand affinity is beneficial to tumor penetration.
However, Lee et al. investigated the distribution of 25 nm epidermal
growth factor receptor (EGFR)-targeted block copolymer micelles and
non-targeted micelles in tumors. The penetration depth of non-targeted
nanomedicine was longer than the active targeting delivery systems
[35]. It might be attributed to the “binding site barrier” effect.
4. Potential drug delivery nanosystems for improving tumor
penetration
4.1. Tumor microenvironment programming
The tumor matrix presents a transport barrier which has significant
negative effect on the penetration of macromolecules within tumor
tissues, thereby limiting the anticancer efficiency of nanoscale delivery
systems. As mentioned above, the tumor extracellular matrix includes
abnormal vascular structures, elevated IFP, tight ECM, CAFs, TAMs, and
hypoxia. It has been demonstrated that the method of regulating the
TME can be a feasible approach to improve the permeability.
4.1.1. Collagenase
Nanomedicine may be entrapped in complex collagen networks
which severely restrict deep penetration. The ECM barriers could be
overcome by collagenase-linked super paramagnetic NPs. There was a
more efficient localization in collagenase- modified NPs in deep tumor
region than non-modified NPs [36]. Goodman et al. systematically as-
sessed the penetration depth of collagenase-coated carboxylate poly-
styrene NPs. The number of collagenase-pretreated nanodrugs which
were delivered to the spheroid core was four times higher than that of
nanoagents with collagenase non-immobilized [37]. In another study,
the pretreatment, which was based on the collagenase liposomes, dis-
assembled the collagen stroma and enhanced tissue permeability into
pancreatic tumors [38]. That is, the NP delivery is significantly pro-
moted through the site-specific degradation of ECM protein.
4.1.2. Hyaluronidase (HAase)
HA (Fig. 2), one of the major ECM elements, binds with collagen to
maintain the integrity of extracellular tumor matrix. The content of HA
in tumor is much higher than that in the normal tissues. This
F. Peng, et al.
Fig. 2. Chemical structure of hyaluronic acid (HA).
phenomenon makes the efficient penetration of NPs difficult. HAase-
modified micelles, which were loaded with photosensitizer chlorine e6
(Ce6), were prepared by Liu et al. to facilitate tumor penetration. It was
found that the conjugation of HAase resulted in a remarkable increase
in perfusion and tumor oxygenation, thus greatly improving the effi-
ciency of photodynamic therapy (PDT) [39]. Moreover, in order to
prolong nanocarrier blood circulation, a relatively low-density poly-
ethylene glycol (PEG) layer was covered in the DOX-loaded PEG-PLGA
NPs. The surface was conjugated with recombinant human hyalur-
onidase PH20 (rHuPH20) for deep tumor penetration in aggressive 4 T1
tumors [40]. In addition, HAase also promoted the development of
nanodrug carriers in convection-enhanced delivery (CED). The tumor
distribution volume of 54 nm monodisperse polystyrene particles was
increased by 64%, which was higher than the control ones without
HAase pre-infusion [41].
4.1.3. Bromelain
Bromelain, a crude enzymatic complex, is one of papain peptidase
family and purified from pineapple stalks [42]. Bromelain-conjugated
mesoporous silica nanoparticle (Br-MSN) was synthesized to increase
nanomedicines uptake in endothelial cells, macrophages and cancer
cells. Since bromelain was able to degrade tumor ECM components, Br-
MSN showed much faster diffusion and digest ability in vitro as well as
in vivo. The bromelain pretreatment extended penetration depth in
matrigel by twofold (from 500 to 1000 μm), thus improving tumor
distribution after injection in 4 T1 tumors [43].
4.1.4. Relaxin
In previous reviews, relaxin, a peptide hormone of insulin family,
had been validated to inhibit collagen production and existing collagen
overexpression. Therapeutic potential of relaxin was tested through
hematopoietic stem cell (HSC)-based approaches. As a result, the pep-
tide hormone relaxin expression decreased collagen amount in the ex-
tracellular matrix to improve the trastuzumab therapy outcome in
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
breast tumors [44]. In the meanwhile, the interstitial transport of
plasmid DNA (pDNA) was deeper after recombinant relaxin treatment
in 4 T1 tumor cell lines. These observations were correlated with the
results from pulsed electric field experiment, enhancing electric field-
mediated gene delivery [45]. Inducement of collagen remolding with
relaxin treatment reduce ECM protein level in collagen-rich tumors and
create more pores for nanomaterials to deeply penetrate tumor stroma.
4.1.5. Losartan
High density of collagen I network prevents the deep tumor pene-
tration of nanomaterials, leading to the impaired anti-tumor efficiency.
Losartan was recognized by Food and Drug Administration (FDA) as
angiotensin II receptor antagonist with notable antifibrotic activity for
hypertension in clinic. It could enhance the penetration and therapeutic
efficiency of nanomedicines [46]. The much better penetration ability
was acquired in the application of losartan-pretreated NP in HSTS26T
and Mu89 mice models, which was explained by the losartan-induced
collagen fiber disruption. Furthermore, the similar efficiency of losartan
had been also revealed in a pioneer study which employed pegylated
liposomal DOX as the drug carrier [47]. In addition, free losartan was
injected before the treatment to decrease the content of collagen.
Afterwards, the pH-responsible liposome was injected to kill cancer
cells. Tumor distribution and target site accumulation were con-
siderably improved by losartan pretreatment [48]. The recent animal
experiment had also clarified the enhanced tumor penetration ability of
losartan-loaded NPs in synergistic of chemotherapy/photo thermal
therapy (PTT) [49].
4.1.6. Regulation of CAFs, TAMs and hypoxia (Table 1)
Based on the abundant CAFs, TAMs in tumors, CAF-targeted lipo-
somes and carboxymethylcellulose NPs were exploited to specifically
eliminate tumor stroma to promote tumor penetration [50–51]. TME
was reprogrammed to favor the delivery of the therapeutic NPs in
desmoplastic cancers [52–53]. The enhanced cancer immunotherapy
was also achieved by the successful TAMs repolarization [54–55]. In
addition, the remission of the hypoxic environment had been confirmed
to achieve deep penetration in the combination of multiple treatments
[56–57].
4.1.7. Growth inhibitors
In normal tissues, angiogenic activators and inhibitors are in dy-
namic equilibrium to ensure blood vasculature growth. However, the
balance is disrupted by the abnormal regulation of growth factors in
pathological circumstances like tumors.
4.1.7.1. Transforming growth factor beta (TGF-β)
inhibitors. Transforming growth factor beta (TGF-β) plays a critical
role during cancer progression regulation. In the later stage of tumor
growth, it acts as a promoter for tumor metastasis. The employment of
short-acting, small molecule TGF-β inhibitors decreased pericyte
coverage of vessels and fibrotic components in the neovasculature of
pancreatic tumors, thus promoting the accumulation of 65 nm micelles
and 90 nm Doxil liposomes [58]. Hence, the introduction of TGF-β
inhibitors was significant to treat intractable solid cancers including
pancreatic adenocarcinoma and diffuse-type gastric cancer. Besides, the
damaged extravasation of 70 nm 1,2-diamino cyclohexane-platinum
(II) (DACHPt) micelles was overcome through administering TGF-β
inhibitors (TGF-β-I) in BxPC3 tumors. Fluorescence microscopic
assessment of BxPC3 tumor pieces confirmed that the accumulation
of 70 nm micelles raised to a comparable level with that of the 30 nm
countparts. The result was consistent with the m-synchrotron radiation
X-ray fluorescence (m-SR-XRF) measurement, which demonstrated the
facilitated intracellular delivery and the enhanced tumoricidal activity
of DACHPt micelles by TGF-β-I [59]. In a study of Huang et al., upon
the down regulation of the Wnt16 (a member of the wingless type
MMTV integration site family) levels by quercetin, targeted lipid/
F. Peng, et al.
calcium/phosphate NPs (LCP-QP NPs) were designed to remodel the
TME to promote the penetration of second-wave NPs into TGF-β
activated NIH3T3 cells [60]. Specifically, the NIH3T3 cells were pre-
treated with 10 μM cisplatin and then activated by 10 ng/mL TGF-β.
The treatment of 10 μM quercetin was confirmed to downregulate
Wnt16 level and then inhibit the effect of TGF-β. This strategy
significantly improved the penetration of cisplatin NPs.
4.1.7.2. Tumor necrosis factor-α (TNF-α) and derivatives. Tumor necrosis
factor-α (TNF-α) played a key role in apoptosis, cell survival and
inflammation. The extravasation of erythrocytes and lymphocytes led
to hemorrhagic necrosis in tumors during isolated limb perfusion (ILP)
cancer treatment [61]. In addition, TNF-α had potential in decreasing
IFP and modulating ECM for better transport [62]. Administration of
TNF-α augmented Doxil regimen rendered the liposomal drugs enter
tumor interstitium, explaining the improved anticancer efficiency [63].
Furthermore, the emergence of TNF derivatives had been already
proved to avoid TNF toxicity and replace TNF for the clinical
application. Vascular targeting with low concentrations of NGR-TNF,
a combination of TNF derivative and CNGRC peptide, was investigated
[64]. It enhanced the tumor penetration of chemical drugs without
obvious toxicity.
4.1.7.3. Platelet-derived growth factor (PDGF) antagonists. Platelet-
derived growth factor (PDGF) receptors are highly expressed in many
tumors, increasing IFP in dermis after anaphylaxis-induced IFP
lowering. One of the most vital reasons leading to elevated IFP and
complex collagen network is the existence of myofibroblasts. PDGF
antagonists decreased the contractile function of new-formed
myofibroblasts in tumor tissues to loosen the ECM [65]. When serine
protease fibroblast activation protein (FAP) on the cell surface was
specifically overexpressed on tumor fibroblasts, the genetic deletion
and pharmacologic inhibition of FAP were investigated to control
myofibroblasts content in tumor ECM. Besides, tumor-associated
fibroblast, the major source of collagen type I, contributed to the
decreased uptake of nanodrugs. Oral DNA vaccines targeting FAP were
constructed to kill tumor-involved fibroblasts. The tumor growth was
markedly suppressed through the decrease of collagen type I content,
opening a new venue in penetration-promoting strategies which
combined immunotherapy with chemotherapy in the future [66].
4.1.7.4. Vascular endothelial growth factor (VEGF) receptor
inhibitors. Vascular endothelial growth factor (VEGF), one of the most
vital angiogenic molecules, is involved with binding to tyrosine kinase
receptors and results in cell proliferation as well as new blood vessel
formation [67]. Therefore, anti-angiogenic therapies which normalize
abnormal tumor vessels can improve the delivery of nanomedicines by
making them less leaky. The method of blocking anti-VEGF- receptor-2
antibody (DC101) had been employed to repair the abnormal
vasculature in mammary tumors, allowing smaller NPs,
macromolecular protein and bovine serum albumin (BSA) to enter
tumors more rapidly. When the size of pores in the vessels were reduced
by the normalization, IFP gradually dropped in the tumor mathematical
model. The limited penetration of larger NPs (125 nm) was attributed
to the increased steric and hydrodynamic hindrance [68]. At present,
anti-VEGF antibodies, such as bevacizumab [69,70], have been used in
the treatment of non-small-cell lung cancer brain metastases,
glioblastomas and triple negative breast cancers.
4.1.7.5. A multikinase inhibitor. Sorafenib (Sf), a multikinase inhibitor,
blocked VEGF receptors and PDGF receptors simultaneously for
advanced hepatocellular carcinoma (HCC) therapy [71]. Sf and VEGF-
siRNA co-loaded liposomes were also constructed for the synergistic
treatment of HCC [72].
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
4.1.8. Soy protein
In addition, different size soy protein NPs could also normalize TME
such as the elevated IFP and higher tumor stress, improving tumor
penetration and impeding tumor growth in living mice. Among dif-
ferent size NPs, DOX-loaded soy protein NPs with the size of 30 nm
exhibited the best cytotoxicity, the best TME modulation efficiency as
well as the deepest penetration depth [73].
4.1.9. Tumor-penetrating peptides
4.1.9.1. iRGD and nRGD. Tumor-specific tissue penetration systems,
which were based on iRGD peptide, were discovered to reverse the
ineffective therapy and drug resistance [74]. iRGD, a type of tumor
penetrating cyclic peptides, harbored the RGD αv-integrin-binding
motif and the RGDK cryptic CendR motif. After being cleaved by cell
surface associated proteases, the CendR motif mediated binding to
neuropilin-1 receptor to achieve efficient tumor penetration [75]. The
combination of active and passive processes extravasated more
efficiently and facilitated the intracellular delivery of cytotoxic
payloads. Poly (epsilon-caprolactone)-b-poly (N-vinylpyrrolidone)
(PCL-b- PVP) NPs were modified with iRGD and exhibited superior
tumor penetration in H22 tumor-bearing mice. However, the mon-
targeted nanomedicines distributed primarily at the periphery of tumor
vessels at 24 h after injection [76]. In addition, the penetration of dual
targeting polymeric NPs, which were functionalized with iRGD and
interleukin-13 peptide, was obviously better than that of interleukin-13
single-modified NPs in C6 cells. It clearly showed that iRGD NP (iRNP)
targeted both tumor neovasculature and glioblastoma multiforme
(GBM) cells through receptor labeling in vitro and in vivo. Similar
iRGD penetration promotion phenomenon had been also reported in the
study of iRGD-decorated prodrug NP self-aggregated hydrogel (PDNP-
gel) [77] and interleukin-24 (IL-24)-iRGD recombinant protein [78]. In
another study, nRGD, which covalently conjugated the alanine-alanine-
asparagine “tail” residual with iRGD peptide, was described to enhance
tumoricidal effect of DOX. The nRGD not only retained tumor
penetration effect of iRGD but also modulated TME with the
depletion of TAMs. Compared with PEG-modified DOX liposomes and
nRGD physical mixtures, the nRGD grafted DOX-loaded liposomes
showed the dramatic improvements in antitumor efficiency [79].
Apart from promoting tumor penetration, the high affinity between
RGD and αv integrin also increased the cell uptake of NPs, which
broadened RGD application in the delivery of biotherapeutics.
4.1.9.2. iNGR. The tumor-penetrating peptide iNGR included three
independent sections: a vascular homing motif, a R/KXXR/K tissue
penetration part and a protease recognition site. It provided a solution
to the poor tumor penetration. It had the dual targeting capacity to
tumor vessels based on CD13 receptors (the receptor of iNGR) and to
tumor cells based on the neuropilin-1 (NRP-1) receptors [80]. Here,
iNGR-modified PEG-PLGA NPs were expected to overcome the leading-
edge difficulty in anti-glioma nanodrug delivery. The acquired iNGR-
NPs were observed to achieve excellent penetration at the glioma sites
by initially binding to amino peptidase N, with iNGR hydrolyzed and
cleaved to CRNGR, and then binding to NRP-1 for the deep penetration.
Convincingly, compared with cNGR-NP, paclitaxel (PTX)-loaded iNGR-
NPs exhibited the increased uptake of human umbilical vein endothelial
cells, thus inducing the strengthened anti-proliferation and anti-tube
formation efficiency with IC50 of 16 ng/mL [81]. Currently, iNGR-
functionalized sterically stabilized liposome (iNGR-SSL) was also
proved to promote the penetration across the tumor vessel wall, the
GBM tissue and further into tumor cells. From the in vivo imaging and
the immunofluorescence staining analysis, the uptake and cytotoxicity
of DOX-loaded iNGR-SSL in HUVEC and U87MG cells were
comparatively higher than that of unmodified SSL/DOX. It was
attributed to the increased tumor accumulation and penetration
mediated by iNGR [82]. In summary, the iNGR modification was an
effective strategy to improve the tumor curative effect and increase the
F. Peng, et al.
survival time of GBM animals.
4.1.10. Sonoporation with microbubbles
Microbubbles are usually filled with hydrophobic gas, which is
coated with 10–100 nm layer made of polymers, proteins, and lipids.
Ultrasound causes microbubble cavitation, i.e., periodically shrink and
expand. It will lead to the formation of temporary pores in the cellular
membrane, increasing permeabilization of surrounding cells to drug
molecules. This process is called sonoporation [83]. The existing re-
search papers verified that ultrasound-activated microbubbles in-
creased the penetration of antitumor drugs for optimal nanoagents
delivery. The combination of DOX and Vevo Micromarker microbubbles
had been evaluated to increase cancer cell death ratio. The improve-
ment was attributed to the deeper penetration of DOX into human U-
87MG glioblastoma and MDA-MB-231 breast cancer cells. Preliminary
data in vitro showed the therapeutical potential in vivo studies [84]. In
the presence of approximately 25 × 106 microbubbles per ml, pulsed
ultrasound exposure enhanced the penetration into the breast cancer
spheroids (300–500 µm diameter) [85]. Therefore, particles with dif-
ferent electrical properties and different particle sizes all achieved
higher penetration. The tumor penetration efficiency, which was pro-
moted by the above microbubbles, was also observed in sonicated tu-
mors to hinder growth [86,87]. And recently, synergistic therapy with
sonoporation effect was developed by DOX-loaded human serum al-
bumin (HSA) NPs and Ce6 encapsulated microbubbles (DOX-NPs/Ce6-
MBs) complex nanosystem (Fig. 3). Through the employment of ultra-
sound, the excellent co-delivery nanocarriers penetrated deeper into
cancerous tumors, exhibiting higher delivery efficiency [88]. As the
Fig. 3. A schematic of efficient delivery of drug and photosensitizer through sonoporation effect. Reprinted with permission from [88].
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
acoustic pressure increased from 1.7 to 6.9 MPa, the penetration depth
of micro-RNA loaded NPs increased by 2-fold into human colon cancer
xenografts [89]. To our knowledge, ultrasound-induced microvesicle
cavitation in Phase I clinical trial was conducted to evaluate the safety
against pancreatic cancers. No additional toxicity and prolonged sur-
vival time were observed. The survival period of patient cohorts ex-
tended from 8.9 to 17.6 months, paving the way towards further clin-
ical researches [90].
4.1.11. Hyperthermia (HT)
Hyperthermia (HT) with thermosensitive delivery systems had been
applied to enhance tumor penetration. After mild HT, we could easily
observe the larger gaps in the endothelial lining of 10 µm and deeper
penetration into the interstitial space to at least 27.5 µm in radius from
the vessel walls [91]. For the more efficacious drug delivery, the
coadministration of DOX thermosensitive liposome (DOX-TSL) and
local HT were characterized to initiate the improvement of tumor
vasculature permeability and TSL extravasation [92]. The effect of DOX
formulation and mild HT were also investigated to enhance penetration
into 2D and 3D cell culture model among multiple human ovarian
cancer cells [93]. Novel temperature-sensitive liposomes were designed
to effectively deliver DOX to tumor nuclei. In vitro results demonstrated
that the local release of DOX from heat-responsive liposomes was ac-
celerated under high intensity focused ultrasound (HIFU). After HIFU-
stimulated heat treatment, anticancer drugs rapidly penetrated into
tumor vascular cells and overcome the barriers of limited penetration.
More efficacious anti-tumor effect in vivo was verified by the 65.2%
tumor inhibition rate in the groups of HIFU-treated mice [94].
F. Peng, et al.
Fig. 4. Chemical structure of acrylate poly (β-amino ester).
Numerical modeling of intraperitoneal (IP) chemotherapy was estab-
lished to examine the impact of HIFU frequency on the penetration
depth of DOX in thermosensitive liposomes. An improvement in drug
penetration depth was observed by changing the HIFU frequency from
0.5 to 1.0 MHz [95]. The phase I study of ultrasound-mediated HT of
lyso-thermosensitive liposomal DOX was perfectly carried out in adult
oncology patients with incurable liver tumors [96].
4.2. Multistage drug delivery platforms
One of successful methods for enhancing drug uptake and pene-
tration is the application of multistage drug delivery platforms. Even
though NPs with larger size were prone to achieve superior accumu-
lation which was gained by EPR effect [97], their penetration had been
also hampered by the physiological barriers in the TME. Even though
NPs with smaller size improved tumor penetration and therapeutic ef-
ficacy, NPs whose size was less than 5 nm were rapidly cleared by
kidney filtration, which resulted in the ineffective killing of cancer cells.
Recently, multistage drug delivery platforms, which could retain com-
paratively large size to ensure the prolonged blood circulation and
switched to smaller particle to promote extravasation, had been envi-
sioned as a promising strategy to meet the contradictory requirements
on particle size. The stimuli utilized to trigger the size alternation of
NPs could be low pH, overexpression of enzymes, glutathione (GSH),
light and chemical reactions.
4.2.1. Low pH
The pH value of normal tissues and blood (~7.4) was higher than
that of TME (~6.5) [98]. The lower pH could be used to trigger the
degradation of acid-cleavable linkers [99–103] or ionization of che-
mical groups for better penetration.
4.2.1.1. The degradation of acid-cleavable linkers in low pH. In a pioneer
research, stimuli-responsive size-switching clustered NPs were prepared
through molecular self-assembly of platinum (Pt) prodrug-conjugated
poly (amidoamine)-graft-poly caprolactone (PCL-CDM-PAMAM/Pt),
PCL and PEG-b -PCL. After accumulating at tumor tissues with lower
pH, 5 nm PAMAM/Pt was discharged from the smart iCluster with
approximately 100 nm via a pH sensitive degradable linker. It also
demonstrated that iCluster significantly improved tumor penetration
and delivered threefold drugs to tumor cells [99]. Kim and colleagues
functionalized the surface of gold NPs (~13 nm) with Bcl-2 antisense
oligodeoxynucleotides (ODNs). Clustering of AuNPs (~150 nm), which
were motivated by the hybridization of i-motif sequence, provided
better tumor targeting efficiency and drug loading capability. Results
showed that apoptosis was accelerated by the disassembly of clustered
AuNPs into single NPs in response to the lower pH [100]. In addition,
the down-regulation of Bcl-2 proteins was observed in the antisense-
grafted size-tunable NPs, which provided multifunctional therapeutic
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
agents in the treatment of tumors. Similar designs were also found in
the study of Sha et al. Size-switching micelle nanocluster (MNC), which
was based on cross-linked and pH-sensitive frameworks between
polyethyleneimine (PEI) and tocopheryl polyethylene glycol succinate
(TPGS), was validated to enhance tumor targeting and penetration
[101]. Moreover, owing to the acid-mediated charge-reversal and
dissociation, the versatile hollow mesoporous silica NPs (HMSNs)
were found to sequentially overcome multiple drug gemcitabine
(GEM) deliver barriers for the enhanced penetration and the
programmed tumor therapy. The enhanced tumor therapy was also
verified in three-dimensional multicellular spheroids and A549 tumor
xenografted mouse model. The well-designed pH-sensitive multistage
polymeric micelleplexes maintained structural stability at physiological
pH (pH 7.4). Once reached to tumor sites, they presented the triggered
release of small PAMAM-Pt dendrimers due to acid-labile characteristic
of the carboxylic amides of CS component (dimethylmaleic anhydride,
DMA), following by the release of cisplatin in intracellular reducing
environment for better chemotherapy efficacy [102].
4.2.1.2. The ionization of chemical groups in low pH. The ultrasensitive
size-tuning systems containing tertiary amine groups had been further
constructed to surmount multiple obstacles in drug delivery. Poly (β-
amino ester) (PAE) (Fig. 4) was a kind of pH-responsive biodegradable
polymers. It was insoluble at higher pH (pH 7.4) and dissolved at a
lower pH due to the protonation of tertiary amino groups. Wang and
colleagues reported a variety of amphiphilic pH-responsive NP
structures including ionizable tertiary amine groups which rapidly
responded to different pH. At neutral pH, these NPs were prepared by
PEG-b-poly (2-azepane ethyl methacrylate)-modified PAMAM
dendrimers (PEG-b-PAEMA-PAMAM) and had an original size of
80 nm for prolonged circulation. When they were transferred to the
acidic TME, the polarity of PAEMA changed from hydrophobic to
hydrophilic, resulting in the instantaneous disintegration into the
dendrimer building blocks and the effective tumor penetration. Both
in vitro and in vivo results showed that pH-triggered size switching was
a viable method to improve drug penetration in the poorly permeable
BxPC-3 pancreatic tumors [10]. Moreover, intelligent size/charge-
changing micellar systems were developed for enhanced drug
delivery. After extravasation from the leakage of tumor vasculatures,
methoxy poly ethylene glycol poly lactide (MPEG-PLA)-PAE micelles
penetrated deeper than MPEG-PLA copolymer micelles. That was
attributed to the protonation of PAE chains in the mildly acidic TME
(Fig. 5). Thus, the occurrence of protonation phenomenon caused size
and charge conversions to increase tumor cellular uptake of curcumin
(CUR) and improve cytoplasmic distribution in human breast cancer
MCF-7 cells [104]. What’s more, the docetaxel (DTX)-loaded MPEG-
PLA-PAE polymeric micelles were manipulated to illustrate the effect of
particle size. When the pH reduced to 6.5 through the addition of
hydrochloric acid (0.1 M), the smaller particles (27.99 nm) percentage
F. Peng, et al.
Fig. 5. A schematic diagram to show the size/charge transformation of MPEG-P LA-PAE micelles in response to the tumor acidic microenvironment. Reprinted with
permission from [104].
increased to 73%, enhancing permeability on 3D KB MCTS model and
showing size-dependent antitumor effect against KB tumors. Taken
together, DTX-pH polymeric micelles (DTX-pHPMs) showed the best
antitumor efficiency with the highest tumor inhibition rate of 84.4% in
oral epidermoid carcinomas [105]. Such size and charge dual-shifting
NPs were also observed in the shell-stacked NPs (SNPs), which
underwent substantial particle size reduction to 40 nm and surface
charge reversal to 8.2 mV for efficient penetration. More importantly,
DOX-loaded NPs penetrated deeper than the nontransformable ones,
showing significant anticancer efficiency and eradicating the
xenografted A549 lung carcinoma cells in mice [106]. Similarly,
deeper penetration and higher tumor suppression rates were obtained
by the disassociation of long-circulating polymer-prodrug hybrid NPs.
The protonation of poly (2-(pentamethyleneimino) ethyl methacrylate)
(PPMEMA) core at pH 6.8 induced the disassociation of long-circulating
polymer-prodrug hybrid NPs to small-size siRNA composites. The
improved tumor penetration was confirmed by the 5-fold higher cell
uptake rate at pH 6.8 than pH 7.4 [107]. Penetration-promoting
phenomenon had been also reported in the study of Wang et al.
When NPs were delivered to the tumor tissues, the inner poly (2-
azepane ethyl methacrylate) (PAEMA) segments converted from
hydrophobic to hydrophilic. The polar transformation of PAEMA was
attributed to the acidic extracellular TME, thereby shrinking micellar
size for the deeper penetration [108].
Another strategy for deep penetration in response to pH variation
was developed by reversibly switchable size nanogels, which were
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
comprised of N-lysinal-N, -succinyl chitosan (NLSC) with protonatable
side groups and poly (N-isopropylacrylamide) (PNIPAM) with BSA
shells. At the pH of 4.5, the protonation caused swelling to 2 µm.
Afterwards, the swollen nanogels caused endo-lysosomal bursting and
drug release. Subsequent shrinking at higher pH enabled transfer to
neighboring tumor cells [109]. These results showed the repeated
swelling-shrinking process ensured the deeper penetration of nanome-
dicines in tumors.
4.2.2. Overexpression of enzymes
Generally, the overexpression of enzymes occurs in a variety of
tumors. This phenomenon has been applied to realize size tuning of NPs
to improve tumor penetration. The enzymes used to trigger the event
can be matrix metalloproteases (MMPs) and hyaluronidase (HAase).
4.2.2.1. Overexpression of matrix metalloproteases (MMPs)
4.2.2.1.1. Gelatin nanoparticles based on the overexpression of
MMPs. Being a substrate of MMP-2, gelatin can be degraded by
MMP-2 and has been employed to construct size tunable NPs. For
example, AuNPs coated gelatin nanoparticles (GNPs) were prepared as
drug delivery systems. The size reduction from 186.5 nm to 59.3 nm in
24 h was observed owing to the presence of MMP-2 (Fig. 6). Therefore,
the obtained G-AuNPs-DOX-PEG with initial larger size possessed better
transvascular and interstitial penetrating effectiveness, showing
admirable antitumor effects in 4 T1 and B16F10 tumors [110]. In
addition, quantum dots (QDs) were assembled onto the surface of GNPs.
F. Peng, et al.
Fig. 6. A) Schematic design procedure of G-AuNPs-DOX-PEG. B) Intracellular delivery process of G-AuNPs-DOX-PEG. Reprinted with permission from [110].
Hybrid nanotherapeutics with a larger size of 100 nm were verified to
exhibit the increased accumulation in the leaky areas of tumor
vasculature due to EPR effect. The shrunken NPs with small size of
10 nm could more readily diffuse throughout the tumor interstitial
regions owing to the gelatin degradation by MMP-2. In vivo study, the
penetration depth of multistage system reached 300 μm from the
injection site, displaying the enhanced tumor extravasation and
homogenous distribution in HT-1080 tumors [111].
Besides, by conjugating the gelatin with dendrigraft poly-lysine
(DGL) and anchoring with angiopep-2 targeting, Gao and co-workers
had fabricated dendrimer-GNPs. After degraded by MMP-2, the particle
size of DOX-DGL-GNPs shrank to 50 nm, showing much wider dis-
tribution of as far as 130 μm from the edge [112]. Moreover, the cell
penetrating peptide TAT and cytotoxic peptide KLAK decorated den-
drimers were synthesized to induce the entrance of nanodrugs into
tumor cells through both membrane and endocytosis routes [113].
MMP2-over expressed TME caused the deprotection of PEG chains,
leading to the obvious decrease of dendrimers diameter and exposure of
KLAK and TAT. It not only improved solid tumor penetrability but also
disrupted mitochondria effectively through the internalization of
bioactive peptides into human primary glioblastoma (U87) tumors.
Further, based on the targeting of lactose [114], through combining
GNPs with small molecule prodrug DOX-Lactose (DOX-Lac), optimized
drug delivery systems (GNPs-DOX-Lac) were developed for enhanced
HCC penetration to overcome a class of pathophysiological barriers
[115].
4.2.2.1.2. MMPs sensitive bond-linked nanoparticles based on the
overexpression of MMPs. The size-switchable systems could be also
constructed by using MMPs sensitive linker to attach smaller particles
onto larger size particles. Herein, MMP-2/pH dual-responsive NPs
(DGL/DOX@NPs) were established to break enzyme-sensitive bonds
in the TME and rapidly release DOX. The process of particle size
alteration rendered the systems take advantage of the enhanced
permeability into both multicellular spheroids (MCSs)and solid
tumors [116]. Analogously, comparing MMP2-sensitive peptide
(PLGLAG) conjugated macromolecules (HA-pep-PAMAM) with MMP2-
insensitive macromolecules (HA-PAMAM), the former could penetrate
tumor tissues due to the degradation by overexpressed MMP-2 in
multicellular spheroids [117]. PLGA-PEG NPs were functionalized
with MMP-2 sensitive linker AGFSGPLGMWSAGSFG. These NPs were
followed by the modification into porous silicon disk to prepare
enzyme-stimulated multistage vector (ESMSV). After intravenously
injected to A375 melanoma lung metastasis bearing mice, more
polymer NPs were released from the ESMSV and 64.6% of the
melanoma cells were polymer NPs positive [118]. As a result, the
enzyme-sensitive size reduction was a reliable strategy to enhance
tumor penetration [119–120].
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
4.2.2.2. Overexpression of hyaluronidase (HAase). Strong cationic
biomaterials possessed several undesirable properties including the
aggregation of erythrocytes and platelets. Hence, Na et al. designed
biodegradable cationic PTX-loaded nanogels (DpNGs), which were
composed of acetylated pullulan (AP) and low molecular weight PEI
(1.8 kDa). The surface of DpNGs was modified by anionic HA. The HA/
DpNG-PTX nanoagents exhibited significant cytotoxicity in
heterogeneous cancer cells via HA degradation by HAase (Fig. 7).
Correspondingly, the IC50 value was 100 times less than free drug and
the invasive distance of HA/DpNG-PTX increased by 2 times than that
of PA-PTX, enhancing the limited tumor penetration [121].
Additionally, Qi laboratory constructed bio-stimuli-sensitive
multistage methotrexate (MTX)-loaded HA NPs which were modified
with PAMAM dendrimers. The initial HA coated PAMAM NPs showed
low cytotoxicity and prolonged systematic circulation. However, they
were easier to be degraded by HAase to release inner PAMAM
dendrimers for deep drug penetration. As expected, the fluorescein
isothiocyanate (FITC) labeled HA/PAMAM NPs penetrated deeply into
A549 3D tumor spheroids [122].
In addition, photothermal therapy (PTT) can kill cancer cells by the
heat generated by photothermal formulations under the irradiation of
near infrared (NIR) light [123,124]. In recent years, many scientists
combined PTT with chemotherapy and gained better achievements. For
example, HAase-motivated size shrinking occurred at the HAase-rich
TME in the intelligent NPs, which were decorated with NIR laser re-
sponsive nitric oxide donor (HN), small size dendrimer prodrug (IDD).
DOX and indocyanine green (ICG) acted as chemotherapy agent and
photothermal agent respectively (Fig. 8). The novel NPs not only in-
duced strong HT effect for PTT but also promoted the NO release to
trigger deep penetration [125]. Moreover, based on the alternation of
the percentage of HA and AuNC@CBSA (small, kidney-clearable, red-
emitting, cationic bovine serum albumin-protected gold nanoclusters),
Gao et al. screened out and evaluated a well-chosen size of 200 nm NPs.
The size-reducible nanoplatform performed excellent combinational
therapy by co-delivering PTX and ICG. It was worth mentioning that HA
could serve both as a shielding agent and a ligand of differentiation 44
(CD44). In the study, HA shielded the NPs with tumor-specific cluster of
CD44 targeting and could be degraded by HAase to reduce size, facil-
itating the penetration effect [126]. HAase-triggered size reduction and
deeper penetration were found in the as-prepared red blood cell (RBC)
membrane-coated biomimetic NPs. HAase specifically hydrolyzed the
HA layers, followed by the exposure of small size gold nanoclusters
(AuNCs) for antitumor combination therapy [127].
Potential drug delivery nanosystems, which are based on the effect
of pH and enzymes, are listed in Table 2.
4.2.3. High-concentration glutathione (GSH)
Photodynamic therapy (PDT) had been tremendously explored for
F. Peng, et al. European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238

Fig. 7. Elucidation of drug delivery procedure of HA/DpNG-PTX. Reprinted with permission from [121].

the treatment of cancer, which was achieved by gathering the photo-
sensitizer (PS) to mediate the incomplete reduction of oxygen upon the
laser light irradiation. High level of reactive oxygen species (ROS) was
generated to induce potent vascular damage and direct tissue destruc-
tion [128]. In normal tissues and cells, the interconversion of glu-
tathione (GSH) and glutathione disulfide (GSSG) ensured cellular redox
homeostasis, which was necessary for cell growth [129]. However, the
GSH concentration range was from 1 × 10-3 to 15 × 10-3 mol/L in
tumor cells, which was significantly higher than normal tissues [130]. It
was attributed to the special requirements of tumor abnormal growth.
Herein, multifunctional nanocomposites had been reported with GSH-
mediated size-tunable characteristic for bioimaging guided synergetic
therapy, including chemotherapy, PDT and Fenton reaction. The initial
size of PEG-decorated up-conversion NPs (UCNPs)-Pt (IV)-ZnFe2O4 was
around 100 nm, which was beneficial for tumor retention. The Pt (IV)
prodrug, which linked UCNPs with ZnFe2O4, was broken owing to
higher GSH in tumor cells. Meanwhile, the nanocomposites were di-
vided into UCNPs (about 25 nm), ZnFe2O4 (about 7 nm) and high

Fig. 8. A) Schematic design procedure of IDDHN. B) Schematic illustration of the synergistic treatments for deep tumor penetration and therapy outcome. Reprinted
with permission from [125].

229
 Table 1
Potential drug delivery nanosystems through regulating CAFs and TAMs and hypoxia.
Factor Mechanism Nanocarrier Active substance Type of tumor Therapy Ref
F. Peng, et al.

CAFs Depletion Liposomal navitoclax DOX Hep G2 Chemotherapy [50]

CAFs Depletion Carboxymethylcellulose NPs DTX PAN 02, OCIP 19, OCIP 23 Chemotherapy [51]
CAFs Reprogram Lipid-coated protamine DNA complexes sTRAIL plasmids UMUC3, NIH3T3 Chemotherapy [52]
CAFs The sensitiveness to FAP-α protein overexpressed Cationic PAMAM dendrimers cross-linked by Asp-Ala-Thr-Gly- Pro-Ala DOX PC-3, CAF Chemotherapy [53]
on CAFs peptide
TAMs Reprogram β-cyclodextrin nanoparticles Resiquimod MC38 Immunotherapy [54]
TAMs Reprogram HA-poly(ethylenimine) (HA-PEI) NPs MicroRNA-125b KRASG12D, P53GEM Immunotherapy [55]
Hypoxia Produce oxygen and relieve hypoxia Albumin–MnO2 NPs Ce6 4 T1 Chemotherapy in conjugation with PDT [56]
Hypoxia Produce oxygen and relieve hypoxia Mesoporous silica nanoparticles Catalase MDA-MB231 Chemotherapy in conjugation with [57]
ultrasound

230
Table 2
Potential drug delivery nanosystems based on the effect of pH and enzymes.
Strategy Carriers Active substance Type of Therapy Ref
tumor

The degradation of acid-cleavable linkers in HMSNs and small-sized Pt- PAMAM GEM A549 Chemotherapy [102]
low pH
The ionization of chemical groups in low pH Polymer − prodrug nanocomplexes SiRNA, MTX MDA-MB-231 Chemotherapy in conjugation with gene [107]
therapy
The ionization of chemical groups in low pH Triblock prodrug polymer micelles Capecitabine 4 T1 Chemotherapy in conjugation with two- [108]
photon AIE imaging
The ionization of chemical groups in low pH Reversible swelling–shrinking nanogel DOX Heps Chemotherapy [109]
Gelatin nanoparticles GNPs and prodrug Dox-Lac complex DOX HepG2 Chemotherapy [115]
MMPs sensitive bond-linked nanoparticles HA-pep-PAMAM macromolecules DOX A549, MCF-7 Chemotherapy [117]
MMPs sensitive bond-linked nanoparticles PEG–MMP2-cleavable peptide–phosphatidylethanolamie (PEG-pp-PE) DOX, dasatinib 4 T1 Chemotherapy [119]
copolymer micelles
MMPs sensitive bond-linked nanoparticles RGD-PEI-SS-PLA/PTX@ MMP-2-sensitive nanoclusters PTX MCF-7 Chemotherapy [120]
Overexpression of hyaluronidase (HAase) Nitric Oxide Donor and HA modified dendrimeric prodrug DOX, ICG 4 T1 Chemotherapy in conjugation with PTT [125]
Overexpression of hyaluronidase (HAase) Red blood cell membrane coated NPs Pheophorbide A, PTX, anti-PD-L1 4 T1 Chemotherapy, PDT and immunotherapy [127]
peptide dPPA
European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
F. Peng, et al.
Fig. 10. Schematic representation of the delivery process of M−DOX / Wtmn (+) to tumor cells. M−Dox / Wtmn (+) was made up of ALK-M−DOX/Wtmn and N3-
M−DO X /Wtmn. Along with the injection of the catalysts in the tumor, the size increased to enhance drug accumulation, thus showing outstanding cytotoxicity as
well as autophagy inhibitory efficiency. Reprinted with permission from [141].
virulent Pt (II). The size transformation had been verified to promote
tumor distribution, penetration. In addition, the core–shell structured
UCNPs- Pt -ZnFe2O4 (UCPZ) -PEG acted as the UV source to motivate
PDT effect and endowed the energy for ZnFe2O4 to yield highly toxic
·OH, taking the place of direct UV illumination. Moreover, based on the
inherent up-conversion luminescence (UCL) and the adopted Yb3+ and
Fe3+, UCPZ-PEG was employed as a multimodality imaging contrast
agent. This research was involved with multiple therapies, including
cancer diagnosis (UCL, X-ray computed tomography (CT)), photo-
acoustic (PA) as well as magnetic resonance imaging (MRI) [131].
4.2.4. Light
4.2.4.1. UV light. Light had been repeatedly utilized to prepare
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Fig. 9. A schematic of DSPE-PEG based
micelles for inhibiting primary tumors
and lymphatic metastasis. Owing to the
small sizes, the drug delivery system
accumulated in the lymph nodes to cure
metastatic tumors. Moreover, copper(I)
triggered CuAAC between azide-func-
tionalized micelles and alkyne-functio-
nalized ones caused the aggregation,
greatly improving tumor accumulation.
Reprinted with permission from [140].
intelligent drug delivery systems. However, the employment of light-
responsive materials of constructing size switchable nanoagents was
rarely reported. Kohane and colleagues described UV light-triggered
size adjustable NPs, which provided spatiotemporal controlled release
of drugs and the enhanced tissue penetration under 365 nm ultraviolet
irradiation. In this system, the spiropyran (SP)-C9 transformed from
hydrophobic cores to zwitterionic merocyanine (MC)-C9 upon UV
irradiation. In the meanwhile, the size of SP-C9 NPs reduced from
150 nm to 40 nm due to the solubility change of SP-C9. ICG-modified
SP NPs penetrated about 8 nm in a collagen gel model without UV
illumination, whereas penetrated 12 nm and 16 nm with 10 and 20 s
irradiation [132]. In a following study, the photoswitchable DTX-
loaded NPs exhibited better therapeutic efficiency than free DTX as
F. Peng, et al.
Fig. 11. Elucidation of the combined strategy: A) losartan pretreatment depleted the collagen I and enhanced penetration of DOX-AuNPs-GNPs. B) MMP2-triggered
size reduction and pH-mediated drug release. Reprinted with permission from [142].
well as DTX-loaded NPs without UV triggering [133]. Another type of
nanoassemblies with light-responsive property was constructed from
linear flexible polyelectrolytes (poly (diallyl dimethylammonium
chloride or quartered poly (4-vinylpyridine)) and ionic photo-
isomerize azo dyes (Acid Yellow 38). After UV irradiation, the
exposure of UV-light caused the size decrease from a hydrodynamic
radius of 94 nm to 62 nm. It was attributed to trans–cis isomerization of
the dyes [134].
4.2.4.2. Near infrared (NIR) light. It was necessary to mention that the
above UV-triggered formulations could really change size, thus
combining the advantages of larger size for extended blood
circulation with smaller size for deep drug penetration. But UV light
was restricted due to the poorer tissue penetration and the damage to
normal tissues [135]. The employment of NIR light with deeper tissue
penetration and higher biocompatibility to control size should be
regarded as a better option. Upon NIR light illumination, the
personalized NIR light-activated UCNPs absorbed NIR light and
selectively released the Pt prodrug complex, which displayed higher
cytotoxicity against human ovarian carcinoma A2780 cells and human
cisplatin-resistant variant A2780cis cells. Besides, the highly activated
caspases effectively cleaved the probe peptide from NP surface, thereby
serving as personalized tumor markers to image in real time in living
cells [136].
4.2.5. Chemical reactions
Breast tumors, melanoma and other malignancies spread through
the lymphatic system to lead to the metastasis [137]. The conventional
nanodrug delivery systems (NDDs) with the fixed size could not address
this dilemma between primary tumor and lymph node metastasis. Not
surprisingly, in Kataoka et al. study, it was verified that smaller NPs
effectively inhibited the growth of lymph node metastasis [138]. Based
on the successful application of Cu (I)-mediated plasmonic nanosensors
in the real clinical practice [139], He et al. designed size-transformable
polymeric micelles for targeted delivery of anti-tumor drugs in primary
tumors and lymph node metastatic tumors. They were modified with
reaction substrate (alkyne group or azide group) on the surface of 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine (D SPE)-PEG micelles
(Fig. 9). The nanocarriers underwent Cu (I) triggered click cycloaddi-
tions: firstly, the small size micelles (~25 nm) easily delivered drugs to
the lymph nodes to cure metastases; secondly, the size increased to
120 nm along with the injection of catalysts (ascorbic acid and copper
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European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
sulfate, where ascorbic acid was used as the reducing agent). The azide
and alkynyl reacted to increase the particle size and promoted tumor
tissue retention for the treatment of primary tumors. As a result, the
tumor lymph node metastasis dropped by 66.7%, showing the deeper
penetration and better inhibitory effect in BALB/C mice [140]. And
recently, similar size-adjustable and azide/alkyne-modified micelles
were employed through Cu (I)-catalyzed click reaction for synergistic
treatment of primary tumor and metastatic cancer (Fig. 10). Un-
doubtedly, through co-delivering DOX and autophagy inhibitor wort-
mannin (Wtmn), the smaller size DSPE-PEG micelles had outstanding
cytotoxicity and obvious suppression effect in melanomas [141]. More
importantly, this novel strategy provided a promising preclinical out-
look for drug delivery to metastatic tumors.
4.3. Combination strategies (Table 3)
Up to now, combination strategies have achieved very good results
in various laboratories. In Gao et al. lab, a dual strategy of adminis-
trating MMP2-sensitive size shrinkable DOX-AuNPs-GNPs with losartan
was elaborated for high intertumoral DOX concentration and high
tumor apoptosis ratio (Fig. 11). Therefore, the combined NPs, in which
the losartan depleted the collagen I, exhibited the optimal drug delivery
efficiency and noticeable penetration efficiency in breast tumor bearing
mice, thus providing a highly attractive way for breast cancer therapy
[142]. In the study of Chen et al., size-changeable “Trojan Horse” na-
nocarriers (THNCs), which were consisted of PTX-loaded Greek soliders
(~20 nm), were assembled in amphiphilic gelatin matrix with the ad-
dition of hydrophilic losartan. As expected, after gelatin was cleaved by
MMP-2, the THNCs exhibited the controllable drug release, higher pe-
netration depth and higher cytotoxicity in 3D tumor spheroid models
[143]. In addition, the enhanced tumor penetration was acquired by
shape transformation as well as RGD, where the shape transformation
was attributed to the overexpression of MMP 9 [144]. Tumor perme-
ability was improved by NIR-induced size shrinkage. UCNPs could
transform NIR to UV–vis and remove PEG for deep penetration [145].
In another study, hydrophobic agents (CPI-613) were conjugated with
hydrophilic polymers via MMP 2 sensitive linker. The simultaneous
targeting of tumor cells and stroma was achieved by the co-delivery of
TGF-β receptor I/II inhibitor and CPI-613 for deeper treatment of
pancreatic cancers [146]. In the study of Jiang et al, TAMs polarization
and acid-triggered size transformation reduced IFP and loosened ECM.
As a result, dandelion-like NPs led to longer circulation and deeper
 Table 3
Examples of combination strategies for improving tumor penetration.
Strategy Drug Type of Tumor Highlight of the research Therapy Ref
F. Peng, et al.

Losartan + gelatin NPs DOX 4 T1 Size-shrinkable gelatin NPs with collagen depletion by losartan Chemotherapy [142]
Losartan + gelatin NPs PTX HeLa Size-changeable gelatin NPs with collagen depletion by losartan Chemotherapy [143]
RGD + MMP-9 sensitive linker DOX H22, 4 T1 NP shape transformation with enhanced penetration by RGD Chemotherapy [144]
NIR + RGD DOX MCF-7 NIR-triggered size shrinkage and RGD activation Chemotherapy [145]
TGF-β receptor I/II inhibitor + MMP-2 sensitive linker CPI-613 PANC-1 Normalization of the stroma and cleavage of MMP-2-responsive linker Chemotherapy [146]
TAMs modulation and low pH PTX 4 T1 Normalization of the stroma and acid-triggered size variation Chemotherapy [147]
CAFs regulation + MMP-2 sensitive linker GEM 4 T1 CAFs-targeted regulation and cleavage of MMP-2-responsive linker Chemotherapy [148]
VEGF inhibitor + low pH Etoposide Luc-A549 The protonation of imidazole group and downregulation of VEGF Chemotherapy [149]
RGD + hyperthermia / 4 T1 Magnetic induction hyperthermia and RGD activation tumor imaging and heat treatment [150]

233
Table 4
Recent and typical clinical therapies.
Pathway Operating conditions Tumors Main conclusion Clinical trial Ref

MMP Inhibitor MMP activity was determined before and after Advanced and refractory solid tumors It is well tolerated and obviously inhibited gelatinase Phase I [151]
S-3304 injection activity in volunteers
Degradation of HA PEGPH20 Metastatic pancreatic ductal Higher drug delivery efficiency than drug alone Randomized phase II [152]
adenocarcinoma study
Losartan Losartan for 8 cycles Locally advanced pancreatic cancer Neoadjuvant therapy with losartan prolonged survival Phase II [153]
rate in patients
Targeting TAMs Emactuzumab every 2 or 3 weeks Advanced/metastatic solid tumors Specific depletion of immunosuppressive macrophages Phase I [154]
VEGF inhibitors Bevacizumab or cetuximab Resected pancreatic carcinoma Feasible and safe Phase II [155]
VEGF inhibitors + TNF-α Lenalidomide Advanced pancreatic cancer Feasible and safe Phase I [156]
TNF-α + NGR A peptide-tumor necrosis factor-a fusion Malignant pleural mesothelioma Stroma normalization and targeted drug deliveryuncit Randomize phase III [64]
protein
Microbubbles in combination with pulsed Acoustic pressure increased from 0.5 to Recurrent GBM Blood-brain barrier disruption and optimal carboplatin Phase 1/2a [157]
ultrasound 1.1 MPa delivery
Hyperthermia Microwave spiral strip applicators operating at Superficial tumors Adjuvant HT improves efficacy in radiation-treated Randomized Trial [158]
433 MHz. patients
European Journal of Pharmaceutics and Biopharmaceutics 151 (2020) 220–238
F. Peng, et al.
tumor penetration in tumor tissues in vivo [147]. Similarly, deep drug
delivery was achieved through CAFs-targeted regulation and the clea-
vage of MMP 2 responsive linker in the treatment of 4 T1 breast cancer
models [148]. Based on the protonation of imidazole groups under
acidic conditions, the co-delivery of VEGF inhibitor and etoposide had
also shown great potential in promoting penetration and inhibiting
tumor proliferation [149]. In a recent study, the penetration depth of
nanoprobes was also promoted by magnetic induction hyperthermia
and RGD activation [150].
4.4. Clinical therapies (Table 4)
At present, penetration-promoting therapies have been put into
clinical trials in cancer treatment. Nevertheless, combination therapies
have been rarely applied to inhibit tumor growth in clinical. In earlier
years, the application of MMP inhibitor and HT was proved to be safe,
effective and well-tolerated for a phase I trial and randomized trial.
Other recent strategies were also shown to optimize drug delivery at
different stages of clinical trials. It turned out that penetration-pro-
moting strategies played a vital role in clinical trials (Table 4).
5. Conclusion and future prospective
In this review, we elaborate the penetration obstacles which exist in
tumor regions, and summarize the important properties of NPs which
hinder deep penetration. Then, the latest and potential drug delivery
nanosystems for improving tumor penetration are analyzed in detail. At
last, recent combination therapies and clinical therapies are introduced
to enhance tumor penetration and antitumor efficiency. These therapies
show great potential in the aspect of chemotherapy, PDT, PTT, bioi-
maging therapy and immunotherapy. However, as our ultimate purpose
is to treat human diseases, the precise doses, suitable time and drug
safety are needed to be highly concerned. In other words, each therapy
requires further testing to define its therapeutic applicability and clin-
ical utility.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (81202480, 81302723); the Natural Science
Foundation of Liaoning Province (2015020749).
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