The second phase, called the maintenance phase, has the aims of keeping the interstitial pressures in balance and optimizing the results obtained from the first phase of the treatment. It consists of using compression garments, skincare, hydration, kinesiotherapy and manual self-massage (Foldi et al., 1985; Camargo & Marx, 2000; Bergmann et al.; Leal et

The aim of using compression garments is to maintain and optimize the results obtained from the first phase of lymphedema treatment and avoid recurrences, through keeping the interstitial pressures in balance. They should be used continuously and only be removed for personal hygiene. The model of garment and the compression used depend on the patient's needs (Fig. 8). Concomitantly with using the compression garments, the patient should also comply with the guidance previously given regarding limb care, hydration and kinesiotherapy. (Foldi et al., 1985; Roucout & Oliveira, 1999; Andersen et al., 2000; Camargo & Marx, 2000; Huit, 2000; Hampton, 2003). In our institution, we use compression garments in conjunction with self-massage and kinesiotherapy in cases of initial lymphedema, with

Lymphatic self-massage, also known as simple manual lymphatic drainage, is a version of manual lymphatic drainage. The technique for this procedure is taught to the patient, who can then perform this alone, at home every day. It involves a series of gentle circular movements that begin with stimulation of the contralateral axillary lymph node chains and the inguinal chains homolateral to the surgical manipulation, followed by gentle movements starting at a place distant from the congested area and moving towards the edematous limb

The patient should begin the self-massage with circular movements in the contralateral axillary region, with the palms in the axilla, lightly and gently moving the skin 30 times, and

(Foldi et al., 1985; Camargo & Marx, 2000; Williams et al., 2002; Linnitt, 2005).

al., 2009; Sarri et al., 2010).

**3.3 Compression garments**

significant improvements achieved.

Fig. 8. Garments and gloves for elastic containment.

**3.4 Lymphatic self-massage**

After hydration, the skin and bone prominences should be protected using cotton gauze and foam, with the aim of filling in any anatomical spaces. The compressive bandaging is done using low-elasticity bandage rolls. It is started on the fingers and then the forearm and the upper arm. The pressure exerted by the bandaging is greater in the distal region and diminishes towards the root of the limb (Fig. 7). It is also important to respect the trophic conditions of the skin. The compressive bandaging is kept in place until the next physiotherapy session, i.e. for two days.

(f) (g)

Fig. 7. Functional compressive bandaging: a) hydration; b) placement of cotton gauze; c) protection with cotton gauze; e) bandaging, starting with the fingers; f) bandaging on forearm; g) bandaging on upper arm.

The second phase, called the maintenance phase, has the aims of keeping the interstitial pressures in balance and optimizing the results obtained from the first phase of the treatment. It consists of using compression garments, skincare, hydration, kinesiotherapy and manual self-massage (Foldi et al., 1985; Camargo & Marx, 2000; Bergmann et al.; Leal et al., 2009; Sarri et al., 2010).

### **3.3 Compression garments**

758 Current Cancer Treatment – Novel Beyond Conventional Approaches

After hydration, the skin and bone prominences should be protected using cotton gauze and foam, with the aim of filling in any anatomical spaces. The compressive bandaging is done using low-elasticity bandage rolls. It is started on the fingers and then the forearm and the upper arm. The pressure exerted by the bandaging is greater in the distal region and diminishes towards the root of the limb (Fig. 7). It is also important to respect the trophic conditions of the skin. The compressive bandaging is kept in place until the next

(a) (b) (c)

(d) (e)

Fig. 7. Functional compressive bandaging: a) hydration; b) placement of cotton gauze; c) protection with cotton gauze; e) bandaging, starting with the fingers; f) bandaging on

(f) (g)

physiotherapy session, i.e. for two days.

forearm; g) bandaging on upper arm.

The aim of using compression garments is to maintain and optimize the results obtained from the first phase of lymphedema treatment and avoid recurrences, through keeping the interstitial pressures in balance. They should be used continuously and only be removed for personal hygiene. The model of garment and the compression used depend on the patient's needs (Fig. 8). Concomitantly with using the compression garments, the patient should also comply with the guidance previously given regarding limb care, hydration and kinesiotherapy. (Foldi et al., 1985; Roucout & Oliveira, 1999; Andersen et al., 2000; Camargo & Marx, 2000; Huit, 2000; Hampton, 2003). In our institution, we use compression garments in conjunction with self-massage and kinesiotherapy in cases of initial lymphedema, with significant improvements achieved.

Fig. 8. Garments and gloves for elastic containment.

#### **3.4 Lymphatic self-massage**

Lymphatic self-massage, also known as simple manual lymphatic drainage, is a version of manual lymphatic drainage. The technique for this procedure is taught to the patient, who can then perform this alone, at home every day. It involves a series of gentle circular movements that begin with stimulation of the contralateral axillary lymph node chains and the inguinal chains homolateral to the surgical manipulation, followed by gentle movements starting at a place distant from the congested area and moving towards the edematous limb (Foldi et al., 1985; Camargo & Marx, 2000; Williams et al., 2002; Linnitt, 2005).

The patient should begin the self-massage with circular movements in the contralateral axillary region, with the palms in the axilla, lightly and gently moving the skin 30 times, and

Evidence-Based Usefulness of Physiotherapy Techniques in Breast Cancer Patients 761

treatment (Fourie & Robb, 2009). It is developed between the first and fifth week after axillary dissection, as tense and painful strings under the skin of the axilla. It may extend as far as the cubital fossa or the medial face of the upper arm (Tilley et al., 2009). It is always associated with pain and limitation of shoulder and elbow movement (Lauridsen et al., 2005). Its incidence and predisposing factors are not well defined in the worldwide literature (Moskovitz et al., 2001). It is believed that interruption of the axillar lymphatic vessels has an important role in the development of this syndrome. Lymphovenous lesions with stasis, thrombophlebitis, aseptic lymphangitis and lesions in the lymphatic ducts also seem to be involved (Johansson et al., 2001; Moskovitz et al., 2001; Lauridsen et al., 2005). The literature is deficient with regard to the approach to be taken in cases of AWS. Some papers have shown that this syndrome resolves within three months, without specific treatment (Moskovitz et al., 2001; Leidenius et al., 2003). Other studies have shown benefits from implementing active range-of-motion exercises, stretching exercises and manual manipulation techniques (Moskovitz et al., 2001; Leidenius et al., 2003; Fourie & Robb, 2009;

In our service, we have used string stretching with very favorable results. The maneuver consists of stretching the string with the thumbs, while applying pressure from central to distal regions, as shown in Fig. 10. This maneuver triggers tolerable local pain that is relieved as soon as the maneuver ends, thereby enabling movements that had previously been limited (Fig. 11). We have also used active and passive kinesiotherapy on the limb and

Fig. 10. Axillary Web Syndrome maneuver: a) string in anterior region of elbow; b) axillary web syndrome maneuver; c) anterior region of forearm after maneuver, without string.

Fig. 11. Axillary Web Syndrome: a) axillary region before the maneuver; b) axillary region after the maneuver; c) anterior region of the elbow before the maneuver; d) anterior region

(a) (b) (c) (d)

Tilley et al., 2009).

light stretching.

of the elbow after the maneuver.

then repeating this in the inguinal region homolateral to the axillary dissection. After the axillary and inguinal lymph node chains have been stimulated, hand movements are made with the aim of shifting the lymph from the operated axilla to the contralateral axilla, in the region above the surgery. The same movement is then made to shift the lymph from the manipulated axilla to the homolateral inguinal region. The movement in each region is repeated 30 times (Fig. 9).

(c) (d)

Fig. 9. Lymphatic self-massage on patient who underwent right axillary dissection: a) axillary lymph node chain stimulation; b) inguinal lymph node chain stimulation; c) wavelike maneuver directing the lymphatic flow towards the contralateral axillary region; d) wavelike maneuver directing the lymphatic flow towards the ipsilateral inguinal region.

#### **3.5 Pneumatic compression**

This consists of mechanical compression with air pumps, on the edematous limb. There are basically two types of compression pumps: the segmental or sequential type and the dynamic type. Individual use of pneumatic compression has not shown satisfactory results, and high incidence of complications has been found. Several studies have combined its use with other types of treatment for lymphedema, such as the use of lymph therapy (Dini et al., 1998; Leduc et al., 1998; Camargo & Marx, 2000; Szuba et al., 2002; Leal et al., 2009).

### **4. Axillary web syndrome**

Axillary web syndrome, also known as cording (Box et al., 2002), axillary strings (Lauridsen et al., 2005), or vascular strings (Johansson et al., 2001), is a sequela of breast cancer

then repeating this in the inguinal region homolateral to the axillary dissection. After the axillary and inguinal lymph node chains have been stimulated, hand movements are made with the aim of shifting the lymph from the operated axilla to the contralateral axilla, in the region above the surgery. The same movement is then made to shift the lymph from the manipulated axilla to the homolateral inguinal region. The movement in each region is

Fig. 9. Lymphatic self-massage on patient who underwent right axillary dissection: a) axillary lymph node chain stimulation; b) inguinal lymph node chain stimulation;

1998; Leduc et al., 1998; Camargo & Marx, 2000; Szuba et al., 2002; Leal et al., 2009).

c) wavelike maneuver directing the lymphatic flow towards the contralateral axillary region; d) wavelike maneuver directing the lymphatic flow towards the ipsilateral inguinal region.

(c) (d)

(a) (b)

This consists of mechanical compression with air pumps, on the edematous limb. There are basically two types of compression pumps: the segmental or sequential type and the dynamic type. Individual use of pneumatic compression has not shown satisfactory results, and high incidence of complications has been found. Several studies have combined its use with other types of treatment for lymphedema, such as the use of lymph therapy (Dini et al.,

Axillary web syndrome, also known as cording (Box et al., 2002), axillary strings (Lauridsen et al., 2005), or vascular strings (Johansson et al., 2001), is a sequela of breast cancer

repeated 30 times (Fig. 9).

**3.5 Pneumatic compression**

**4. Axillary web syndrome**

treatment (Fourie & Robb, 2009). It is developed between the first and fifth week after axillary dissection, as tense and painful strings under the skin of the axilla. It may extend as far as the cubital fossa or the medial face of the upper arm (Tilley et al., 2009). It is always associated with pain and limitation of shoulder and elbow movement (Lauridsen et al., 2005). Its incidence and predisposing factors are not well defined in the worldwide literature (Moskovitz et al., 2001). It is believed that interruption of the axillar lymphatic vessels has an important role in the development of this syndrome. Lymphovenous lesions with stasis, thrombophlebitis, aseptic lymphangitis and lesions in the lymphatic ducts also seem to be involved (Johansson et al., 2001; Moskovitz et al., 2001; Lauridsen et al., 2005). The literature is deficient with regard to the approach to be taken in cases of AWS. Some papers have shown that this syndrome resolves within three months, without specific treatment (Moskovitz et al., 2001; Leidenius et al., 2003). Other studies have shown benefits from implementing active range-of-motion exercises, stretching exercises and manual manipulation techniques (Moskovitz et al., 2001; Leidenius et al., 2003; Fourie & Robb, 2009; Tilley et al., 2009).

In our service, we have used string stretching with very favorable results. The maneuver consists of stretching the string with the thumbs, while applying pressure from central to distal regions, as shown in Fig. 10. This maneuver triggers tolerable local pain that is relieved as soon as the maneuver ends, thereby enabling movements that had previously been limited (Fig. 11). We have also used active and passive kinesiotherapy on the limb and light stretching.

Fig. 10. Axillary Web Syndrome maneuver: a) string in anterior region of elbow; b) axillary web syndrome maneuver; c) anterior region of forearm after maneuver, without string.

Fig. 11. Axillary Web Syndrome: a) axillary region before the maneuver; b) axillary region after the maneuver; c) anterior region of the elbow before the maneuver; d) anterior region of the elbow after the maneuver.

Evidence-Based Usefulness of Physiotherapy Techniques in Breast Cancer Patients 763

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## **5. Limitation of shoulder movement**

Restriction of shoulder movement, which is one of the complications following axillary lymph node dissection, may occur because of tissue and nerve lesions, with prevalence of 7- 36%. This musculoskeletal disorder of the shoulder results in considerable joint debility and pain. The symptoms generally diminish within three months, but they may become chronic, thus interfering with these patients' quality of life (Kärki et al., 2001; Beurskens et al., 2007). Early physiotherapeutic treatment is effective for this disorder and promotes faster functional recovery (Kärki et al., 2001; Lauridsen et al., 2005; Beurskens et al., 2007). The treatment should be progressive, taking care in manipulating the limb so as to avoid tissue or muscle injuries. Passive exercises, assisted active exercises, active exercises of the scapular belt, neck relaxation and postural guidance should be undertaken.

## **6. Painful post-mastectomy syndrome**

Chronic pain secondary to surgical treatment for breast cancer is of neuropathic origin or results from muscle and ligament injuries. Intercostal brachial neuralgia has been correlated most frequently in axillary treatments, in which the nerve may become injured because of its proximity (Poleshuck et al., 2006; Labreze et al., 2007; Couceiro et al., 2009). Such pain may be continuous or intermittent, with varying intensity, and it can be located in the anterior wall of the chest, the axilla or the medial face of the upper arm (Vecht et al., 1989; Caffo et al., 2003; Burckhardt & Jones, 2005). The physiotherapeutic treatment consists of specific analgesia techniques, such as transcutaneous electrical nerve stimulation (TENS), cryotherapy and kinesiotherapy. It can also be implemented in association with the use of analgesic medications.

## **7. Conclusion**

In conclusion, physiotherapeutic interventions on patients who have undergone axillary lymph node manipulation can be implemented at any postoperative stage. However, it is increasingly certain that early intervention significantly minimizes the emergence of lymphedema.

## **8. Acknowledgements**

The authors thank Barretos Cancer Hospital, and their coworkers Thiago Buosi and Carla Laurienzo da Cunha Andrade.

### **9. References**

Restriction of shoulder movement, which is one of the complications following axillary lymph node dissection, may occur because of tissue and nerve lesions, with prevalence of 7- 36%. This musculoskeletal disorder of the shoulder results in considerable joint debility and pain. The symptoms generally diminish within three months, but they may become chronic, thus interfering with these patients' quality of life (Kärki et al., 2001; Beurskens et al., 2007). Early physiotherapeutic treatment is effective for this disorder and promotes faster functional recovery (Kärki et al., 2001; Lauridsen et al., 2005; Beurskens et al., 2007). The treatment should be progressive, taking care in manipulating the limb so as to avoid tissue or muscle injuries. Passive exercises, assisted active exercises, active exercises of the scapular

Chronic pain secondary to surgical treatment for breast cancer is of neuropathic origin or results from muscle and ligament injuries. Intercostal brachial neuralgia has been correlated most frequently in axillary treatments, in which the nerve may become injured because of its proximity (Poleshuck et al., 2006; Labreze et al., 2007; Couceiro et al., 2009). Such pain may be continuous or intermittent, with varying intensity, and it can be located in the anterior wall of the chest, the axilla or the medial face of the upper arm (Vecht et al., 1989; Caffo et al., 2003; Burckhardt & Jones, 2005). The physiotherapeutic treatment consists of specific analgesia techniques, such as transcutaneous electrical nerve stimulation (TENS), cryotherapy and kinesiotherapy. It can also be implemented in association with the use of

In conclusion, physiotherapeutic interventions on patients who have undergone axillary lymph node manipulation can be implemented at any postoperative stage. However, it is increasingly certain that early intervention significantly minimizes the emergence of

The authors thank Barretos Cancer Hospital, and their coworkers Thiago Buosi and Carla

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**5. Limitation of shoulder movement**

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analgesic medications.

**8. Acknowledgements**

Laurienzo da Cunha Andrade.

**7. Conclusion**

lymphedema.

**9. References**

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**32**

*1Mexico 2USA*

**Negative Impact of Paclitaxel Crystallization on**

Paclitaxel, better known as Taxol® its original name and now trade mark, and its direct derivatives, has been one of the most effective drugs used against tumors and cancer (Rowinsky et al., 1990; Rowinsky & Donehower, 1995) due to its ability to stabilize microtubules in the mitotic spindle and thus stopping the cell cycle in eukaryotic cells (Wani et al.,1971). The stabilization of microtubules is due to the specific bond between Taxol® and beta tubulin preventing microtubule depolymerization (Schiff et al., 1979). It has also been reported that Taxol® inhibits the process of angiogenesis (Wang et al., 2003). These Taxol® qualities have been taken as an advantage for the usage of this drug in killing carcinogenic

One of the main drawbacks of this molecule is its high hydrophobicity; making it practically insoluble in water which hinders their use in treatments inside the human body. In order to overcome such inconveniences, several strategies have been developed such as the use of adjuvants like Cremofor/Etanol (CrEL), solubilizant agent that facilitates the intravenously administration of the drug, process termed as chemotherapy. This technique has brought many disadvantages such as the fact of reducing considerably the drug's diffusion capacity, making that a limited quantity of the drug be reached into the target site (Marupudi et al., 2007), and therefore limiting the concentration level needed to eliminate tumors. To solve these inconveniences it has been resolved to increase the delivery cycles. The usage of Taxol® in CrEL and the high doses have produced undesired consequences with a numerous of hypersensitivity reactions and side effects including nausea, vomiting, urticaria, abdominal pain, diaphoresis, and other (Weiss et al., 1990; Wiernik et al., 1987). Looking for minimizing such secondary reactions, new options in the field of drug delivery are being explored. In the last decade a variety of drug delivery systems have been proposed as carriers of hydrophobic anticancer drugs. The list of these systems includes

**1. Introduction**

cells in tumors.

**Hydrogels and Novel Approaches for**

*1Materials Science and Engineering, Institute of Engineering and Technology,*

**Anticancer Drug Delivery Systems**

Javier S. Castro1,2, Lillian V.Tapia1, Rocio A. Silveyra1,

Carlos A. Martinez1 and Pierre A. Deymier2

*University of Arizona, Tucson AZ 85721*

*Autonomous University of Juarez City, Juarez City, Chih. 2Department of Materials Science and Engineering,*

## **Negative Impact of Paclitaxel Crystallization on Hydrogels and Novel Approaches for Anticancer Drug Delivery Systems**

Javier S. Castro1,2, Lillian V.Tapia1, Rocio A. Silveyra1,

Carlos A. Martinez1 and Pierre A. Deymier2

*1Materials Science and Engineering, Institute of Engineering and Technology, Autonomous University of Juarez City, Juarez City, Chih. 2Department of Materials Science and Engineering, University of Arizona, Tucson AZ 85721 1Mexico 2USA*

#### **1. Introduction**

766 Current Cancer Treatment – Novel Beyond Conventional Approaches

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Sakorafas, G. H., Peros, G., Cataliotti, L. & Vlastos, G. (2006). Lymphedema following

Sarri, A. J., Moriguchi, S. M., Dias, R., Perez, S. V., Silva, E. T., Koga, K. H., Mathes, A. G. Z.,

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Tilley, A., Thomas-Maclean, R. & Kwan, W. (2009). Lymphatic cording or axillary web

Van Der Veen, P., De Voogdt, N., Lievens, P., Duquet, W., Lamote, J. & Sacre, R. (2004).

resection. *Lymphology*, Vol. 37, No. 4, Dec, pp. 206-8, ISSN 0024-7766 (Print) Vecht, C. J., Van De Brand, H. J. & Wajer, O. J. (1989). Post-axillary dissection pain in breast

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Williams, A. F., Vadgama, A., Franks, P. J. & Mortimer, P. S. (2002). A randomized

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pp. 254-61, ISSN 0961-5423 (Print)

axillary lymph node dissection for breast cancer. *Surg Oncol*, Vol. 15, No. 3, Nov,

Santos, M. J., Rocha, E. T. & Haikel, R. L. (2010). Physiotherapeutic stimulation: Early prevention of lymphedema following axillary lymph node dissection for breast cancer treatment. *Experimental and Therapeutic Medicine*, Vol. 1, pp. 147-152,

W. & Danoff, J. (2010). Pre-operative assessment enables early diagnosis and recovery of shoulder function in patients with breast cancer. *Breast Cancer Res Treat*, Vol. 120, No. 1, Feb, pp. 135-47, ISSN 1573-7217 (Electronic) ISSN 0167-6806

advances in breast cancer-related lymphedema of the arm: lymphatic pump failure and predisposing factors. *Lymphat Res Biol*, Vol. 7, No. 1, pp. 29-45, ISSN 1539-6851

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Vol. 49, pp. 11-15, ISSN 0103-5894

(Linking)

(Print)

Paclitaxel, better known as Taxol® its original name and now trade mark, and its direct derivatives, has been one of the most effective drugs used against tumors and cancer (Rowinsky et al., 1990; Rowinsky & Donehower, 1995) due to its ability to stabilize microtubules in the mitotic spindle and thus stopping the cell cycle in eukaryotic cells (Wani et al.,1971). The stabilization of microtubules is due to the specific bond between Taxol® and beta tubulin preventing microtubule depolymerization (Schiff et al., 1979). It has also been reported that Taxol® inhibits the process of angiogenesis (Wang et al., 2003). These Taxol® qualities have been taken as an advantage for the usage of this drug in killing carcinogenic cells in tumors.

One of the main drawbacks of this molecule is its high hydrophobicity; making it practically insoluble in water which hinders their use in treatments inside the human body. In order to overcome such inconveniences, several strategies have been developed such as the use of adjuvants like Cremofor/Etanol (CrEL), solubilizant agent that facilitates the intravenously administration of the drug, process termed as chemotherapy. This technique has brought many disadvantages such as the fact of reducing considerably the drug's diffusion capacity, making that a limited quantity of the drug be reached into the target site (Marupudi et al., 2007), and therefore limiting the concentration level needed to eliminate tumors. To solve these inconveniences it has been resolved to increase the delivery cycles. The usage of Taxol® in CrEL and the high doses have produced undesired consequences with a numerous of hypersensitivity reactions and side effects including nausea, vomiting, urticaria, abdominal pain, diaphoresis, and other (Weiss et al., 1990; Wiernik et al., 1987). Looking for minimizing such secondary reactions, new options in the field of drug delivery are being explored. In the last decade a variety of drug delivery systems have been proposed as carriers of hydrophobic anticancer drugs. The list of these systems includes

Negative Impact of Paclitaxel Crystallization on Hydrogels

al., 2007).

**2.2 Paclitaxel crystallization in hydrogels**

target. a) Bar 2.5 µm, b) bar 10 µm.

and Novel Approaches for Anticancer Drug Delivery Systems 769

there are hydrogels that exhibit the phenomenon of reverse thermal gelation, also known as thermally induced hydrogels. In this case they are liquid at lower temperatures, but they solidify when their temperature rises (Klouda & Mikos, 2008). This property has been exploited to create hydrogels that remain liquid at temperatures below 30 ° C, but they solidify at temperatures close to physiological temperature (37 °C). Therefore, they are better injected subcutaneously as a liquid and solidify when they reach body temperature. Some of these hydrogels are tri-block copolymer synthesized by open-ring polymerization such as poly (ethylene glycol)-poly(epsilon-caprolactone)-poly (ethylene glycol) (PEG-PCL-PEG) (ChangYang et al., 2009; Yu, 2009), tri-block copolymer poly(ethylene oxide)-poly (oxidopropileno) -poly (ethylene oxide) (PEO-PPO-PEO) (Li & Li, 2008), and biopolymer chitosan (C) neutralized with b-glycerophosphate (GP) (Ruel-Gariépy et al., 2004). Most of these hydrogels are studied for drug delivery and some have already been used successfully for this purpose. For example, PEO-PPO-PEO-name Poloxamer 407 has been used to prolong the release of lidocaine (Chen et al., 2004). Also, some natural polymers exhibit the phenomenon of reverse thermal gelation such as those based on chitosan, which have reported positive results; for instance, a solution of chitosan with 40% weight of PEG has been tested on the release of serum bovine holding a sustained release of at least 70 hours (Bhattarai et al., 2005). Other organic hydrogels with this property are hydroxypropyl

cellulose (Cai et al., 2003; Uraki et al., 2004) and methyl cellulose (Kumar et al., 1993).

Among the new methods aimed to replace intravenous chemotherapy for the treatment of tumors there are certain proposals which emphasize the use of temperature-sensitive hydrogels loaded with anticancer drugs. They suggest the injection of the thermosensitive hydrogels containing drug directly into the affected area (tumor), due to its reverse thermal behavior it solidifies inside the body and then the drug is released gradually. The most common proposed systems for Taxol® delivery have been those that offer direct incorporation of the drug into the thermosensitive hydrogel (Chun et al., 2009; Dai et al., 2006; Kasala et al., 2008; Livnat et al., 2005; Marupudi et al., 2007; Ruel-Gariépy et al., 2004; Shi & Burt, 2004; Woo Sun et al., 2007). Just to mention some examples, the alginate hydrogel with polyethylene glycol (PEG) (Livnat et al., 2005), the hydrogel Dx-g-PCL result of the synthesis of polycaprolactone (PLC) and Dextran (Dextran70 and Dextran 500) (Shi & Burt, 2004), so as the thermosensitive chitosan-based hydrogel BST-Gel liquid (Marupudi et

We have studied Taxol® crystallization in aqueous solutions and hydrogels (Castro et al., 2010). In both environments Taxol® forms needle-like crystals that grow concentrically forming complex morphologies such as spherulites, in the case of heterogeneous nucleation, or sheaves (axialites) when they are formed by homogeneous nucleation. In solution or gel these crystals can be observed by DIC (Differential Interference Contrast) microscopy or by fluorescent microscopy when they are labeled with a fluorescent dye such as rhodamine, as described in (Castro et al. 2009). Dried they can be analyzed by scanning electron microscopy (Figure 2). solution by heterogeneous nucleation with a Taxol concentration of 50 µM. Sample was prepared by drying a drop in a TEM (Transmission Electron Microscope) cooper grid and then metalized by sputter coating with a gold-palladium

drug loaded hydrogels, drug loaded nanoparticles, functionalized nanoparticles and some combined systems. Through this chapter we review the current trends for the transport and delivery of the most common anticancer drugs. Based in facts we point a delicate issue, Taxol crystallization in hydrogels and its negative impact, and finally we make a discussion about novel approaches for drug delivery against cancer.

## **2. Hydrogels for drug delivery systems**

Currently the development of smart hydrogels that can respond to external stimuli such as variations in temperature, pH, and electric fields or hydrogels with controlled biodegradability has been used in biomedical applications. As such hydrogels are used in a wide range of applications including tissue engineering and regenerative medicine (Lee & Mooney, 2001), diagnosis (Van Der Linden et al., 2003), cell immobilization (Jen et al., 1996), bimolecular and cell separation (Wang et al., 1993), barriers to regulate the biological adhesion (Bennett et al., 2003). Hydrogels can be made, in theory, from any water-soluble polymer, encompassing a wide range of chemical compositions and physical properties. The polymer forms aggregates that form a three-dimensional matrix with interconnected pores in which the solvent (usually water or aqueous solutions) and other particles can diffuse (Figure 1). Some hydrogels have a high capacity to contain compounds that can be released in a controlled manner for therapeutic purposes. Its porosity allows the loading of drugs within the polymer matrix and their subsequent release at a rate that depends on the coefficient of diffusion of drug through the gel matrix in the case of hydrophilic drugs. For hydrophobic drugs, the rate of release depends on the rate of degradation of the gel. Besides their biodegradability can be designed to be via enzymatic, hydrolytic, or environmental (pH, temperature or electric fields).

Fig. 1. Hydrogel structures observed by FESEM (Field Emission Scanning Electron Microscope). a) Chitosan-Glycerophosphate termosensitive hydrogel. Bar 40µm. b) Agarose hydrogel. Bar 250 nm.

#### **2.1 Thermosensitive hydrogels for anticancer drug delivery**

Commonly hydrogels gelatinize when their temperature drops; in other words they are present in a liquid state at higher temperatures, and they solidify when it drops. However,

drug loaded hydrogels, drug loaded nanoparticles, functionalized nanoparticles and some combined systems. Through this chapter we review the current trends for the transport and delivery of the most common anticancer drugs. Based in facts we point a delicate issue, Taxol crystallization in hydrogels and its negative impact, and finally we make a discussion

Currently the development of smart hydrogels that can respond to external stimuli such as variations in temperature, pH, and electric fields or hydrogels with controlled biodegradability has been used in biomedical applications. As such hydrogels are used in a wide range of applications including tissue engineering and regenerative medicine (Lee & Mooney, 2001), diagnosis (Van Der Linden et al., 2003), cell immobilization (Jen et al., 1996), bimolecular and cell separation (Wang et al., 1993), barriers to regulate the biological adhesion (Bennett et al., 2003). Hydrogels can be made, in theory, from any water-soluble polymer, encompassing a wide range of chemical compositions and physical properties. The polymer forms aggregates that form a three-dimensional matrix with interconnected pores in which the solvent (usually water or aqueous solutions) and other particles can diffuse (Figure 1). Some hydrogels have a high capacity to contain compounds that can be released in a controlled manner for therapeutic purposes. Its porosity allows the loading of drugs within the polymer matrix and their subsequent release at a rate that depends on the coefficient of diffusion of drug through the gel matrix in the case of hydrophilic drugs. For hydrophobic drugs, the rate of release depends on the rate of degradation of the gel. Besides their biodegradability can be designed to be via enzymatic, hydrolytic, or environmental

Fig. 1. Hydrogel structures observed by FESEM (Field Emission Scanning Electron

**2.1 Thermosensitive hydrogels for anticancer drug delivery**

Microscope). a) Chitosan-Glycerophosphate termosensitive hydrogel. Bar 40µm. b) Agarose

Commonly hydrogels gelatinize when their temperature drops; in other words they are present in a liquid state at higher temperatures, and they solidify when it drops. However,

about novel approaches for drug delivery against cancer.

**2. Hydrogels for drug delivery systems**

(pH, temperature or electric fields).

hydrogel. Bar 250 nm.

there are hydrogels that exhibit the phenomenon of reverse thermal gelation, also known as thermally induced hydrogels. In this case they are liquid at lower temperatures, but they solidify when their temperature rises (Klouda & Mikos, 2008). This property has been exploited to create hydrogels that remain liquid at temperatures below 30 ° C, but they solidify at temperatures close to physiological temperature (37 °C). Therefore, they are better injected subcutaneously as a liquid and solidify when they reach body temperature. Some of these hydrogels are tri-block copolymer synthesized by open-ring polymerization such as poly (ethylene glycol)-poly(epsilon-caprolactone)-poly (ethylene glycol) (PEG-PCL-PEG) (ChangYang et al., 2009; Yu, 2009), tri-block copolymer poly(ethylene oxide)-poly (oxidopropileno) -poly (ethylene oxide) (PEO-PPO-PEO) (Li & Li, 2008), and biopolymer chitosan (C) neutralized with b-glycerophosphate (GP) (Ruel-Gariépy et al., 2004). Most of these hydrogels are studied for drug delivery and some have already been used successfully for this purpose. For example, PEO-PPO-PEO-name Poloxamer 407 has been used to prolong the release of lidocaine (Chen et al., 2004). Also, some natural polymers exhibit the phenomenon of reverse thermal gelation such as those based on chitosan, which have reported positive results; for instance, a solution of chitosan with 40% weight of PEG has been tested on the release of serum bovine holding a sustained release of at least 70 hours (Bhattarai et al., 2005). Other organic hydrogels with this property are hydroxypropyl cellulose (Cai et al., 2003; Uraki et al., 2004) and methyl cellulose (Kumar et al., 1993).

Among the new methods aimed to replace intravenous chemotherapy for the treatment of tumors there are certain proposals which emphasize the use of temperature-sensitive hydrogels loaded with anticancer drugs. They suggest the injection of the thermosensitive hydrogels containing drug directly into the affected area (tumor), due to its reverse thermal behavior it solidifies inside the body and then the drug is released gradually. The most common proposed systems for Taxol® delivery have been those that offer direct incorporation of the drug into the thermosensitive hydrogel (Chun et al., 2009; Dai et al., 2006; Kasala et al., 2008; Livnat et al., 2005; Marupudi et al., 2007; Ruel-Gariépy et al., 2004; Shi & Burt, 2004; Woo Sun et al., 2007). Just to mention some examples, the alginate hydrogel with polyethylene glycol (PEG) (Livnat et al., 2005), the hydrogel Dx-g-PCL result of the synthesis of polycaprolactone (PLC) and Dextran (Dextran70 and Dextran 500) (Shi & Burt, 2004), so as the thermosensitive chitosan-based hydrogel BST-Gel liquid (Marupudi et al., 2007).

#### **2.2 Paclitaxel crystallization in hydrogels**

We have studied Taxol® crystallization in aqueous solutions and hydrogels (Castro et al., 2010). In both environments Taxol® forms needle-like crystals that grow concentrically forming complex morphologies such as spherulites, in the case of heterogeneous nucleation, or sheaves (axialites) when they are formed by homogeneous nucleation. In solution or gel these crystals can be observed by DIC (Differential Interference Contrast) microscopy or by fluorescent microscopy when they are labeled with a fluorescent dye such as rhodamine, as described in (Castro et al. 2009). Dried they can be analyzed by scanning electron microscopy (Figure 2). solution by heterogeneous nucleation with a Taxol concentration of 50 µM. Sample was prepared by drying a drop in a TEM (Transmission Electron Microscope) cooper grid and then metalized by sputter coating with a gold-palladium target. a) Bar 2.5 µm, b) bar 10 µm.

Negative Impact of Paclitaxel Crystallization on Hydrogels

and require major rethinking.

and Novel Approaches for Anticancer Drug Delivery Systems 771

Direct incorporation of paclitaxel into hydrogels can lead to crystallization of the drug which can dramatically decrease the therapeutic effects and effectiveness of paclitaxel delivery systems. Supporting our theory, we find in the work of Chun (2009) who reported that *in vivo* experiments with rats, contrary to what would be expected, the hydrogel with a lower concentration of Taxol® was more effective at tumor inhibition than hydrogel with the highest concentration. Although the authors did not provide explanations for such unexpected results, for us a good reason of this fact is the phenomenon of Taxol® crystallization, which is enhanced at higher concentrations. All this experimental evidence could be enough reason to believe that these systems do not meet the desired expectations

Fig. 3. Taxol crystals formed in hydrogels. a) in aqueous solution, b) in agarose hydrogel, c) in poly (L-lactic acid) hydrogel, d) Chitosan-Glycerophosphate hydrogel. All samples

In order to minimize the inconvenient of the hydrophobicity many authors have proposed the encapsulation of hydrophobic drugs in nanoparticles, this would allow increasing the amount of drug administered without using nasty solvents such as CrEL in the case of paclitaxel. Thus it is being explored new types of delivery systems based in nanocarriers such as micelles (You et al., 2008), nanoparticles (Liang et al., 2006), thin films (Shi & Burt, 2004), and microspheres (Liu et al., 2007) among others (Ferrari & Downing, 2005), whose effectiveness is related to its size, modification of pharmacokinetics, biodistribution,

prepared at a paclitaxel concentration of 100 µM.

**3. Nanoparticles for drug delivery**

The process of crystallization in aqueous solutions and hydrogels is quite similar and we can describe it in few words, Taxol crystallization follows the classical homogeneous nucleation theory. For low supersaturation (low Taxol concentrations), only a small number of crystals nuclei may form whereas for high supersaturation (>20 μM) the number of nuclei increases drastically with increasing Taxol® concentration.

Fig. 2. Taxol crystals observed by FESEM. These crystals were obtained in aqueous solution.

As we mentioned before, the most common Hydrogel-based Taxol® delivery systems reported in literature suggest the direct incorporation of the drug into the thermosensitive hydrogel (Chun et al., 2009; Dai et al., 2006; Kasala et al., 2008; Livnat et al., 2005; Marupudi et al., 2007; Ruel-Gariépy et al., 2004; Shi & Burt, 2004; Woo Sun et al., 2007). But nobody, except Shi & Burt (2004), has reported Taxol crystallizacion in their systems. We claim that this phenomenon is present in most of these works, unfortunately this problem has been a neglected topic by many researchers and may go unnoticed because Taxol® crystals are not detectable by commonly used techniques for the characterization of the hydrogels. Perhaps, researchers have been concerned primarily in designing a good delivery system, neglecting other aspects of the drug, such as its crystallization. In studies reported in the literature that follow this line, no one takes into account, neither they mentioned nor reported studies of crystallization of Taxol® in hydrogels, despite that the use of the drug concentrations is greatly exceeded in its limit of solubility in aqueous solutions (0.77μM ) (Shi & Burt 2004).

In order to provide evidence of Taxol crystallization in these hydrogels we have performed several experiments following similar conditions to that reported in the papers mentioned before. For instance we used similar hydrogels such as agarose, chitosan, poly (L-lactic acid) PLLA and thermosensitive Chitosan-Glycerophosphate and we also used Taxol concentrations similar, or even lower, to that reported in that works. As result we have found that Taxol® crystallization is always present in hydrogels and thermosensitive hydrogels (Figure 3). According to our observations Taxol® crystallization follows the behavior of the classical homogenous nucleation theory, as mentioned before. That means that at low Taxol concentrations (<30 µM) few but big (15-25 µm) axialites are present, while at higher many and smaller axialites are observed (Castro et al., 2011). Moreover paclitaxel crystals are very stable in aqueous environments. We have observed these crystals up to two months of formation without any noticeable change. Hence we can speculate that Taxol crystals inside the body probably would last for long time activating immune reactions.

The process of crystallization in aqueous solutions and hydrogels is quite similar and we can describe it in few words, Taxol crystallization follows the classical homogeneous nucleation theory. For low supersaturation (low Taxol concentrations), only a small number of crystals nuclei may form whereas for high supersaturation (>20 μM) the number of nuclei

Fig. 2. Taxol crystals observed by FESEM. These crystals were obtained in aqueous solution. As we mentioned before, the most common Hydrogel-based Taxol® delivery systems reported in literature suggest the direct incorporation of the drug into the thermosensitive hydrogel (Chun et al., 2009; Dai et al., 2006; Kasala et al., 2008; Livnat et al., 2005; Marupudi et al., 2007; Ruel-Gariépy et al., 2004; Shi & Burt, 2004; Woo Sun et al., 2007). But nobody, except Shi & Burt (2004), has reported Taxol crystallizacion in their systems. We claim that this phenomenon is present in most of these works, unfortunately this problem has been a neglected topic by many researchers and may go unnoticed because Taxol® crystals are not detectable by commonly used techniques for the characterization of the hydrogels. Perhaps, researchers have been concerned primarily in designing a good delivery system, neglecting other aspects of the drug, such as its crystallization. In studies reported in the literature that follow this line, no one takes into account, neither they mentioned nor reported studies of crystallization of Taxol® in hydrogels, despite that the use of the drug concentrations is greatly exceeded in its limit of solubility in aqueous solutions (0.77μM ) (Shi & Burt 2004). In order to provide evidence of Taxol crystallization in these hydrogels we have performed several experiments following similar conditions to that reported in the papers mentioned before. For instance we used similar hydrogels such as agarose, chitosan, poly (L-lactic acid) PLLA and thermosensitive Chitosan-Glycerophosphate and we also used Taxol concentrations similar, or even lower, to that reported in that works. As result we have found that Taxol® crystallization is always present in hydrogels and thermosensitive hydrogels (Figure 3). According to our observations Taxol® crystallization follows the behavior of the classical homogenous nucleation theory, as mentioned before. That means that at low Taxol concentrations (<30 µM) few but big (15-25 µm) axialites are present, while at higher many and smaller axialites are observed (Castro et al., 2011). Moreover paclitaxel crystals are very stable in aqueous environments. We have observed these crystals up to two months of formation without any noticeable change. Hence we can speculate that Taxol crystals inside the body probably would last for long time activating immune reactions.

increases drastically with increasing Taxol® concentration.

Direct incorporation of paclitaxel into hydrogels can lead to crystallization of the drug which can dramatically decrease the therapeutic effects and effectiveness of paclitaxel delivery systems. Supporting our theory, we find in the work of Chun (2009) who reported that *in vivo* experiments with rats, contrary to what would be expected, the hydrogel with a lower concentration of Taxol® was more effective at tumor inhibition than hydrogel with the highest concentration. Although the authors did not provide explanations for such unexpected results, for us a good reason of this fact is the phenomenon of Taxol® crystallization, which is enhanced at higher concentrations. All this experimental evidence could be enough reason to believe that these systems do not meet the desired expectations and require major rethinking.

Fig. 3. Taxol crystals formed in hydrogels. a) in aqueous solution, b) in agarose hydrogel, c) in poly (L-lactic acid) hydrogel, d) Chitosan-Glycerophosphate hydrogel. All samples prepared at a paclitaxel concentration of 100 µM.

## **3. Nanoparticles for drug delivery**

In order to minimize the inconvenient of the hydrophobicity many authors have proposed the encapsulation of hydrophobic drugs in nanoparticles, this would allow increasing the amount of drug administered without using nasty solvents such as CrEL in the case of paclitaxel. Thus it is being explored new types of delivery systems based in nanocarriers such as micelles (You et al., 2008), nanoparticles (Liang et al., 2006), thin films (Shi & Burt, 2004), and microspheres (Liu et al., 2007) among others (Ferrari & Downing, 2005), whose effectiveness is related to its size, modification of pharmacokinetics, biodistribution,

Negative Impact of Paclitaxel Crystallization on Hydrogels

hydroxifenil) retinamide (Tomoyuki et al., 2008).

that the nanoparticles contain a highly cytotoxic drug.

the drug.

and Novel Approaches for Anticancer Drug Delivery Systems 773

The hydrophobic core also has other important features such as protecting the drug from being deactivated by enzymes or other bioactive species from the aqueous medium (blood fluid) (Wilhelm et al., 1991; (Yokoyama et al., 1990). Also, this medium affects the rate of drug release, in many cases declining it, as the release rate is controlled both by the stability of the micelles as the hydrophobicity of their core and the chemical species used to attach the drug to the polymer backbone. These factors may be independent of the properties of

While each system has its advantages and disadvantages, micelles and nanospheres formed by amphiphilic copolymers have greater flexibility in their synthesis and uses, and they have had good results with hydrophobic drugs. The difference lies in the method of preparation and length of the hydrophobic segment. Although for some authors the nanospheres are systems that perform in vivo by presenting better retention properties of the drug, they have a burst effect when the drug is readily released; in this sense, the nanospheres are overcome by the micelles who exhibit more stable behaviors (Gaucher et al., 2010; Kwon, 2003). We can mention some examples of micelles made of different polymers used to carry drugs. We cite a few micelles synthesized from copolymer PEG-PASP-DMEDA loaded with ammonium glycyrrhizate (AMG) poorly water soluble drug used against Hepatitis C (Yang et al., 2009). Copolymer polylactide / polyethylene glycol is synthesized by polymerization of ring-opened was used to encapsulate paclitaxel without the presence of an organic solvent (Kim et al., 2009). Also, some micelles are sensitive to changes in pH, this feature can be used to control drug release as self-assembled micelles of copolymer PEG-b-PMA with divalent metals loaded with doxorucibin (Li et al., 2009). Tamoxifen and paclitaxel have also been charged in micelles with a hydrophilic block of poly [2 - (ethyl methacriloyloxi) phosphorylcholine] (MPC), hydrophobic block and a pH sensitive poly [2 - (diisopropylamine)ethyl methacrylate] (DPA) (FA-MPC-DPA) (Licciardi et al., 2008). Doxorubicin was loaded using thermosensitive blocks such as poly (Nisopropylacrylamide-co-N, N-dimethyl-acrylamide) in which the hydrophobic part was synthesized from poly (D, L-lactide), poly(epsilon-caprolactone) or poly (D, L-lactide-co-ԑcaprolactone) (Masamichi et al., 2006). Another type of thermosensitive micelles have been synthesized from poly (N-isopropylacrylamide) and poly (butyl methacrylate) also loaded with doxorubicin (Chung et al., 1999). Similarly, micelles have been developed and modified in the copolymer as synthesized from poly (ethylene glycol)-poly (aspartateester groups heptyl, nonyl, phenyl propyl benzyl and for greater stabilization of the drug N-(4 -

Encapsulation of paclitaxel in nanoparticles is very promising and results have been favorable, judging by the reports in the literature. However, most authors suggest the use of nanoparticles to be injected into the bloodstream, following the route of traditional chemotherapy, which somehow still has the disadvantage of low specificity in which the problem is attacked. But that's not all, perhaps the main problem is not the lack of specificity of the method, but the risk involved in the use of nanoparticles in the bloodstream. Currently, no one knows for sure the adverse effects of the use of nanoparticles in the bloodstream, since its size could pass biological membranes or barriers and it has unknown effects on areas of the body, becoming a health risk factor , as noted by experts on the subject of the risks of nanotechnology in medicine (Scott et al., 2008). To this, we must add

delivery control and toxicity reduction (Jun et al., 2008). Each one of these particles has unique characteristics that should be carefully analyzed taking into account the type of drug and the desired interaction to obtain the adequate system delivery. The liposome usage for delivery agent of the anticancer drugs is very broad, such as Doxil®, this drug is commercially distributed and used in several ovarian cancer treatments (Gordon et al., 2001). However, the liposomes present several disadvantages due to their high permeability and stability *in vivo* (Conlin et al., 2009). Dendrimers are macromolecules characterized by their structural monodispersivity and symmetry. They consist of a central nucleus and its diverse ramifications. It contains different functional groups generally located on the exterior, which play an important role in their properties (Caminade et al., 2005). A disadvantage is its difficulty to be synthesized (Tomalia et al., 1990). The dendrimers are used in research studies for the prevention of sexually transmitted diseases (Zolnik & Sadrieh, 2009), as contrasting agents in Magnetic Resonance Imaging (Zolnik & Sadrieh, 2009), combined with anticancer drugs (Malik et al., 1999), among other usages. The nanocapsules are vesicular systems with colloidal size, in which the drug is located inside and surrounded by a polymeric membrane (Soppimath et al., 2001). In this system, the nucleus consists of oily liquid and a simple polymeric layer covers it, this system has shown to be effective in encapsulating and releasing certain types of hydrophobic drugs (Ameller et al., 2003). Nanospheres are solid colloidal particles in which the drug can be dissolved, encapsulated, conjugated or absorbed (Gref et al., 1995; Soppimath et al., 2001). They are generally larger than micelles and although its elimination is slow due to its hydrophobicity, are susceptible to renal filtration and the mono nuclear phagocyte system (MPS), It is therefore necessary to modify its surface (Letchford & Burt, 2007). Good stability has been achieved in nanospheres using amphiphilic copolymers (Gref et al., 1995), so it is possible the encapsulation of the bioactive molecule at the core of the nanosphere (Yanasarn et al., 2009), for example, it has been observed that the nanoparticles synthesized from emulsion of lecithin / water loaded with paclitaxel are more effective in attacking cancer cells than pure paclitaxel (Hu et al., 2009). In another study, gold nanoparticles encapsulated in nanospheres of chitosan and poly (acrylic acid) CS-PAA-Au have been used as delivery system drugs as well as for observation of cells (Latere et al., 2002).

Within this range of particles could not miss the micelles, which have had a great interest in the issue of drug delivery and location into tumors (Lee et al., 2003; Yokoyama et al., 1999). Micelles are amphiphilic molecules consisting of hydrophilic and hydrophobic segments. Its outer layer, which is highly hydrated, provides stability in aqueous environments, while the hydrophobic core allows the incorporation of water-insoluble drugs. Moreover, if the polymeric micelles have a diameter ranging from 10 to 100nm allows long circulation in the bloodstream avoiding defenses of the mono nuclear phagocyte system and suppressing renal clearance. They can be large enough to avoid being excreted by the kidneys but small enough to pass the interendothelial cell filtration (Yang et al., 2009).

As a result, drug activity continues after a single application over a long period of time. In addition, these micelles remain intact at levels below the critical micelle concentration (CMC), so they retain their structure and preferentially accumulate in solid tumors via the permeability and retention effect (EPR) (Nakayama et al., 2007; Saez et al., 2004). Its amphiphilic nature gives them a central place in the area of the release of hydrophobic drugs, due to its excellent drug storage capacity in its core.

delivery control and toxicity reduction (Jun et al., 2008). Each one of these particles has unique characteristics that should be carefully analyzed taking into account the type of drug and the desired interaction to obtain the adequate system delivery. The liposome usage for delivery agent of the anticancer drugs is very broad, such as Doxil®, this drug is commercially distributed and used in several ovarian cancer treatments (Gordon et al., 2001). However, the liposomes present several disadvantages due to their high permeability and stability *in vivo* (Conlin et al., 2009). Dendrimers are macromolecules characterized by their structural monodispersivity and symmetry. They consist of a central nucleus and its diverse ramifications. It contains different functional groups generally located on the exterior, which play an important role in their properties (Caminade et al., 2005). A disadvantage is its difficulty to be synthesized (Tomalia et al., 1990). The dendrimers are used in research studies for the prevention of sexually transmitted diseases (Zolnik & Sadrieh, 2009), as contrasting agents in Magnetic Resonance Imaging (Zolnik & Sadrieh, 2009), combined with anticancer drugs (Malik et al., 1999), among other usages. The nanocapsules are vesicular systems with colloidal size, in which the drug is located inside and surrounded by a polymeric membrane (Soppimath et al., 2001). In this system, the nucleus consists of oily liquid and a simple polymeric layer covers it, this system has shown to be effective in encapsulating and releasing certain types of hydrophobic drugs (Ameller et al., 2003). Nanospheres are solid colloidal particles in which the drug can be dissolved, encapsulated, conjugated or absorbed (Gref et al., 1995; Soppimath et al., 2001). They are generally larger than micelles and although its elimination is slow due to its hydrophobicity, are susceptible to renal filtration and the mono nuclear phagocyte system (MPS), It is therefore necessary to modify its surface (Letchford & Burt, 2007). Good stability has been achieved in nanospheres using amphiphilic copolymers (Gref et al., 1995), so it is possible the encapsulation of the bioactive molecule at the core of the nanosphere (Yanasarn et al., 2009), for example, it has been observed that the nanoparticles synthesized from emulsion of lecithin / water loaded with paclitaxel are more effective in attacking cancer cells than pure paclitaxel (Hu et al., 2009). In another study, gold nanoparticles encapsulated in nanospheres of chitosan and poly (acrylic acid) CS-PAA-Au have been used as delivery

system drugs as well as for observation of cells (Latere et al., 2002).

enough to pass the interendothelial cell filtration (Yang et al., 2009).

drugs, due to its excellent drug storage capacity in its core.

Within this range of particles could not miss the micelles, which have had a great interest in the issue of drug delivery and location into tumors (Lee et al., 2003; Yokoyama et al., 1999). Micelles are amphiphilic molecules consisting of hydrophilic and hydrophobic segments. Its outer layer, which is highly hydrated, provides stability in aqueous environments, while the hydrophobic core allows the incorporation of water-insoluble drugs. Moreover, if the polymeric micelles have a diameter ranging from 10 to 100nm allows long circulation in the bloodstream avoiding defenses of the mono nuclear phagocyte system and suppressing renal clearance. They can be large enough to avoid being excreted by the kidneys but small

As a result, drug activity continues after a single application over a long period of time. In addition, these micelles remain intact at levels below the critical micelle concentration (CMC), so they retain their structure and preferentially accumulate in solid tumors via the permeability and retention effect (EPR) (Nakayama et al., 2007; Saez et al., 2004). Its amphiphilic nature gives them a central place in the area of the release of hydrophobic The hydrophobic core also has other important features such as protecting the drug from being deactivated by enzymes or other bioactive species from the aqueous medium (blood fluid) (Wilhelm et al., 1991; (Yokoyama et al., 1990). Also, this medium affects the rate of drug release, in many cases declining it, as the release rate is controlled both by the stability of the micelles as the hydrophobicity of their core and the chemical species used to attach the drug to the polymer backbone. These factors may be independent of the properties of the drug.

While each system has its advantages and disadvantages, micelles and nanospheres formed by amphiphilic copolymers have greater flexibility in their synthesis and uses, and they have had good results with hydrophobic drugs. The difference lies in the method of preparation and length of the hydrophobic segment. Although for some authors the nanospheres are systems that perform in vivo by presenting better retention properties of the drug, they have a burst effect when the drug is readily released; in this sense, the nanospheres are overcome by the micelles who exhibit more stable behaviors (Gaucher et al., 2010; Kwon, 2003). We can mention some examples of micelles made of different polymers used to carry drugs. We cite a few micelles synthesized from copolymer PEG-PASP-DMEDA loaded with ammonium glycyrrhizate (AMG) poorly water soluble drug used against Hepatitis C (Yang et al., 2009). Copolymer polylactide / polyethylene glycol is synthesized by polymerization of ring-opened was used to encapsulate paclitaxel without the presence of an organic solvent (Kim et al., 2009). Also, some micelles are sensitive to changes in pH, this feature can be used to control drug release as self-assembled micelles of copolymer PEG-b-PMA with divalent metals loaded with doxorucibin (Li et al., 2009). Tamoxifen and paclitaxel have also been charged in micelles with a hydrophilic block of poly [2 - (ethyl methacriloyloxi) phosphorylcholine] (MPC), hydrophobic block and a pH sensitive poly [2 - (diisopropylamine)ethyl methacrylate] (DPA) (FA-MPC-DPA) (Licciardi et al., 2008). Doxorubicin was loaded using thermosensitive blocks such as poly (Nisopropylacrylamide-co-N, N-dimethyl-acrylamide) in which the hydrophobic part was synthesized from poly (D, L-lactide), poly(epsilon-caprolactone) or poly (D, L-lactide-co-ԑcaprolactone) (Masamichi et al., 2006). Another type of thermosensitive micelles have been synthesized from poly (N-isopropylacrylamide) and poly (butyl methacrylate) also loaded with doxorubicin (Chung et al., 1999). Similarly, micelles have been developed and modified in the copolymer as synthesized from poly (ethylene glycol)-poly (aspartateester groups heptyl, nonyl, phenyl propyl benzyl and for greater stabilization of the drug N-(4 hydroxifenil) retinamide (Tomoyuki et al., 2008).

Encapsulation of paclitaxel in nanoparticles is very promising and results have been favorable, judging by the reports in the literature. However, most authors suggest the use of nanoparticles to be injected into the bloodstream, following the route of traditional chemotherapy, which somehow still has the disadvantage of low specificity in which the problem is attacked. But that's not all, perhaps the main problem is not the lack of specificity of the method, but the risk involved in the use of nanoparticles in the bloodstream. Currently, no one knows for sure the adverse effects of the use of nanoparticles in the bloodstream, since its size could pass biological membranes or barriers and it has unknown effects on areas of the body, becoming a health risk factor , as noted by experts on the subject of the risks of nanotechnology in medicine (Scott et al., 2008). To this, we must add that the nanoparticles contain a highly cytotoxic drug.

Negative Impact of Paclitaxel Crystallization on Hydrogels

presenting a universal binding system.

for study.

**5. Conclusions**

and Novel Approaches for Anticancer Drug Delivery Systems 775

oligonucleotides that can identify a large number of specific molecules (Torchilin et al., 2003; Tuerkand & Gold, 1990). We have seen that PEG-PLA micelles with an RNA aptamer specifically bind to an antigen on the membrane of prostate tumor (PSMA). Nanoparticles with the induction of aptamer showed 77 times more specificity to PSMA receptor than nonfunctionalized particles (Ellingtonand & Szostak, 1990). The use of ligands has yielded promising results for the location of cancer cells. This will allow drug release systems based on nanoparticles using different ligands that recognize different types of cancer. There is further research to look for future and further specific treatments with low or no side effects. Instead of using a single ligand, nanoparticles' specificity can be potentiated using multiple ligands in a single particle. Sooner or later, these systems will have the ability to attack different cancers at once or they will be designed to exchange specific ligands in an easy way, having a generic nanoparticle system capable of receiving any desired ligand

There are few studies that have integrated particles or micelles loaded with Taxol® in thermosensitive hydrogels. As good examples we can mention the system proposed by Jiang Liu et al. (2007) (Liu et al., 2007). This system consists of a biodegradable gelatin sponge containing PLGA-PTX microspheres (polylactide-co-glycolic acid-paclitaxel) in order to provide continuous local release of PTX (paclitaxel). They obtained a more prolonged release rate for up to 19 days, in contrast to simple systems where the paclitaxel was released within a range less than one hour. In another study presented by Yang Yang et al. (2009) joined Docetaxol (DTX) in a pluronic gel F127(PF127) injectable thermosensitive mixed micelles prepared with the same material (PF127) and loaded with Taxol® (system GMM). During a test of 156 hours, it was found that the proposed system maintains a

prolonged release of DTX compared with other control systems (Yang et al., 2009).

The development of combined drug delivery systems based on efficient and innovative drug encapsulation in functionalized nanoparticles to detect and attack cancer cells, using thermosensitive hydrogels as vehicles that can be injected locally can be an excellent option for the treatment of cancerous tumors, thereby eliminating the supply of intravenous drugs and consequently the severe side effects. Specifically for Taxol®, its encapsulation in nanoparticles would prevent crystallization when deposited in the hydrogel. In addition, the release rates could be controlled in a double form, both by the action of the degradation of gel particles released and at the same time by the degradation of the particle containing the Taxol®. Achieving prolonged drug release would increase the therapeutic effects for longer periods. An added value to these systems is the funcionalization or even multifuncionalization of nanoparticles with ligands or antibodies for high specificity to cancer cells, which should increase the efficiency and the therapeutic effectiveness of the system. Even today very few researchers have worked with Taxol® delivery systems with the characteristics listed above; making this theme of the project attractive and innovative

Researchers are currently exploring new methods for transport and controlled release of drugs for cancer treatment, thereby seeking to reduce the side effects the of currently

**4. Combined systems based on hydrogels and nanoparticles**

#### **3.1 Functionalization of nanoparticles**

Particle functionalization with specific ligands for cancer cells has gained considerable attention for those looking for more specificity in their systems carrying the drug. Work is being currently done to prove their effectiveness with different ligand-receptor systems in a variety of cancer cell lines to test the efficiency of the functionalization of these systems. The effectiveness of the functionalization of nanoparticles lies in the premise of the existence of certain molecules in tumors are different from those found in normal tissues and can be identified as biomarkers of tumorogenesis (Mohd & Mohammad, 2009). Depending on its test site, they may be biomarkers of tissue or circulatory. Tissue biomarkers have different categories such as membrane receptors, oncogenes, tumor suppressor genes, nucleic antigens, growth factors and components of degradation. Circulatory biomarkers include a broad category of tumor-associated antigens (TAA). Selective biomarkers can identify risks and help tumor detection, and an early diagnosis allows appropriate therapeutic interventions for an effective treatment. In addition to these biomarkers, it is also common to find an over expression of certain proteins membrane in cancer cells, which has been used for the use of specific ligands or molecules to these proteins, such as folic acid to folate receptors, to obtain greater interaction of nanoparticles to cancer cells. In this case, the effectiveness of the functionalization is more a matter of statistics than specificity by itself, since having the over expression of these receptors will increase the likelihood of attracting a greater number of drug-loaded nanoparticles to diseased cells.

Micelles have been recently reported to have been functionalized with folic acid as a specific ligand , which is strongly attracted to its receptor in the cell membrane. The folate receptor is a protein that is generated in large quantities during cell proliferation in several types of cancer cells such as ovarian, breast, brain and lung (Ross et al., 1994).

Also, peptides have been used for the functionalization of micelles as locators of cancer cells. The nature of polypeptides allows performance optimization of specific ligands by adjusting the sequence or conformation of the peptides. One example is the peptide cRGD (cyclic Arg-Gly-Asp-D-Phe-Lys) specific for αvβ3 receptor produced in large amounts in tumor endothelial cells (Nasongkla et al., 2004). These receptors are a cell membrane protein that is affected with the growth of tumors, local invasiveness and potentially in metastasis, but it is not detected in quiescent vessels (Rueg et al., 2002; Teti et al., 2002; Vamer & Cheresh, 1996). This membrane receptor increases its levels in vascular angiogenesis, thus making tumor treatment specific for the generation of new blood vessels (Wermuth et al., 1997). Carbohydrate functionalized micelles, asialoglycoprotein receptor (ASGPR) is a recipient of lecithin membrane commonly found in liver cells (Ashwelland & Harford, 1982). In hepatocellular carcinoma are high levels of (ASGPR), which helps the specificity of chemotherapy for liver (Wands & Blum, 1991). Carbohydrate molecules such as galactose and mannose are specific ligands of this receptor (Goto et al., 1994; Jansen et al., 1991).

The use of antibodies as specific ligands is promising as they are able to bind to a range of specific antigens in cancer cells. The combination of a brain-specific antibody increased to 5 fold the neuroleptic action in charged micelles than in non-functionalized micelles, and it is 20 times greater than for the free drug (Kabanov et al., 1989). Micelles have been reported (PEG-PE) functionalized with 2 antibodies, a monoclonal anti-cancer antibody (mAb2C5) and anti-myosin (mAb2G4). Both antibodies have great ability to bind to the substrates after conjugation of the micelles. The antibody 2C5 in micelles loaded with paclitaxel increased 4 times the drug accumulation in the tumor after 2 hours (Torchilin et al., 2003). Furthermore, there is the functionalization of micelles with aptamers. The aptamers are DNA and RNA

Particle functionalization with specific ligands for cancer cells has gained considerable attention for those looking for more specificity in their systems carrying the drug. Work is being currently done to prove their effectiveness with different ligand-receptor systems in a variety of cancer cell lines to test the efficiency of the functionalization of these systems. The effectiveness of the functionalization of nanoparticles lies in the premise of the existence of certain molecules in tumors are different from those found in normal tissues and can be identified as biomarkers of tumorogenesis (Mohd & Mohammad, 2009). Depending on its test site, they may be biomarkers of tissue or circulatory. Tissue biomarkers have different categories such as membrane receptors, oncogenes, tumor suppressor genes, nucleic antigens, growth factors and components of degradation. Circulatory biomarkers include a broad category of tumor-associated antigens (TAA). Selective biomarkers can identify risks and help tumor detection, and an early diagnosis allows appropriate therapeutic interventions for an effective treatment. In addition to these biomarkers, it is also common to find an over expression of certain proteins membrane in cancer cells, which has been used for the use of specific ligands or molecules to these proteins, such as folic acid to folate receptors, to obtain greater interaction of nanoparticles to cancer cells. In this case, the effectiveness of the functionalization is more a matter of statistics than specificity by itself, since having the over expression of these receptors will increase the likelihood of attracting

Micelles have been recently reported to have been functionalized with folic acid as a specific ligand , which is strongly attracted to its receptor in the cell membrane. The folate receptor is a protein that is generated in large quantities during cell proliferation in several types of

Also, peptides have been used for the functionalization of micelles as locators of cancer cells. The nature of polypeptides allows performance optimization of specific ligands by adjusting the sequence or conformation of the peptides. One example is the peptide cRGD (cyclic Arg-Gly-Asp-D-Phe-Lys) specific for αvβ3 receptor produced in large amounts in tumor endothelial cells (Nasongkla et al., 2004). These receptors are a cell membrane protein that is affected with the growth of tumors, local invasiveness and potentially in metastasis, but it is not detected in quiescent vessels (Rueg et al., 2002; Teti et al., 2002; Vamer & Cheresh, 1996). This membrane receptor increases its levels in vascular angiogenesis, thus making tumor treatment specific for the generation of new blood vessels (Wermuth et al., 1997). Carbohydrate functionalized micelles, asialoglycoprotein receptor (ASGPR) is a recipient of lecithin membrane commonly found in liver cells (Ashwelland & Harford, 1982). In hepatocellular carcinoma are high levels of (ASGPR), which helps the specificity of chemotherapy for liver (Wands & Blum, 1991). Carbohydrate molecules such as galactose and mannose are specific ligands of this receptor (Goto et al., 1994; Jansen et al., 1991). The use of antibodies as specific ligands is promising as they are able to bind to a range of specific antigens in cancer cells. The combination of a brain-specific antibody increased to 5 fold the neuroleptic action in charged micelles than in non-functionalized micelles, and it is 20 times greater than for the free drug (Kabanov et al., 1989). Micelles have been reported (PEG-PE) functionalized with 2 antibodies, a monoclonal anti-cancer antibody (mAb2C5) and anti-myosin (mAb2G4). Both antibodies have great ability to bind to the substrates after conjugation of the micelles. The antibody 2C5 in micelles loaded with paclitaxel increased 4 times the drug accumulation in the tumor after 2 hours (Torchilin et al., 2003). Furthermore, there is the functionalization of micelles with aptamers. The aptamers are DNA and RNA

**3.1 Functionalization of nanoparticles**

a greater number of drug-loaded nanoparticles to diseased cells.

cancer cells such as ovarian, breast, brain and lung (Ross et al., 1994).

oligonucleotides that can identify a large number of specific molecules (Torchilin et al., 2003; Tuerkand & Gold, 1990). We have seen that PEG-PLA micelles with an RNA aptamer specifically bind to an antigen on the membrane of prostate tumor (PSMA). Nanoparticles with the induction of aptamer showed 77 times more specificity to PSMA receptor than nonfunctionalized particles (Ellingtonand & Szostak, 1990). The use of ligands has yielded promising results for the location of cancer cells. This will allow drug release systems based on nanoparticles using different ligands that recognize different types of cancer. There is further research to look for future and further specific treatments with low or no side effects. Instead of using a single ligand, nanoparticles' specificity can be potentiated using multiple ligands in a single particle. Sooner or later, these systems will have the ability to attack different cancers at once or they will be designed to exchange specific ligands in an easy way, having a generic nanoparticle system capable of receiving any desired ligand presenting a universal binding system.

## **4. Combined systems based on hydrogels and nanoparticles**

There are few studies that have integrated particles or micelles loaded with Taxol® in thermosensitive hydrogels. As good examples we can mention the system proposed by Jiang Liu et al. (2007) (Liu et al., 2007). This system consists of a biodegradable gelatin sponge containing PLGA-PTX microspheres (polylactide-co-glycolic acid-paclitaxel) in order to provide continuous local release of PTX (paclitaxel). They obtained a more prolonged release rate for up to 19 days, in contrast to simple systems where the paclitaxel was released within a range less than one hour. In another study presented by Yang Yang et al. (2009) joined Docetaxol (DTX) in a pluronic gel F127(PF127) injectable thermosensitive mixed micelles prepared with the same material (PF127) and loaded with Taxol® (system GMM). During a test of 156 hours, it was found that the proposed system maintains a prolonged release of DTX compared with other control systems (Yang et al., 2009).

The development of combined drug delivery systems based on efficient and innovative drug encapsulation in functionalized nanoparticles to detect and attack cancer cells, using thermosensitive hydrogels as vehicles that can be injected locally can be an excellent option for the treatment of cancerous tumors, thereby eliminating the supply of intravenous drugs and consequently the severe side effects. Specifically for Taxol®, its encapsulation in nanoparticles would prevent crystallization when deposited in the hydrogel. In addition, the release rates could be controlled in a double form, both by the action of the degradation of gel particles released and at the same time by the degradation of the particle containing the Taxol®. Achieving prolonged drug release would increase the therapeutic effects for longer periods. An added value to these systems is the funcionalization or even multifuncionalization of nanoparticles with ligands or antibodies for high specificity to cancer cells, which should increase the efficiency and the therapeutic effectiveness of the system. Even today very few researchers have worked with Taxol® delivery systems with the characteristics listed above; making this theme of the project attractive and innovative for study.

## **5. Conclusions**

Researchers are currently exploring new methods for transport and controlled release of drugs for cancer treatment, thereby seeking to reduce the side effects the of currently

Negative Impact of Paclitaxel Crystallization on Hydrogels

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applied chemotherapy as well as looking for battling more efficiently and accurately such disease. Although there are a number of published research focused on this issue, the fact is that so far no systems have emerged to prove conclusively its efficacy, although always positive progress has been shown, each proposed system suffers from certain drawbacks that slows down the progress towards real applications. The disadvantages found in the systems of transport and release of anticancer drugs proposed in recent years can be overcome by taking advantage of each method and combining them into drug delivery systems composed with more sophistication and complexity than their predecessors. This idea has already begun to be implemented by some researchers who have integrated drugloaded nanoparticles on thermosensitive hydrogels (Liu et al., 2007), but these authors have not exploited the alternative of functionalization or multi-functionalization of nanoparticles. So, there is still much room for much improvement in these drug delivery systems and it is where researchers could find novel ideas. For example we can think in to make functionalized or multifunctionalized nanoparticles loaded with a anticancer drug, then incorporate them into a thermosensitive hydrogel. With this proposed system, the hydrogel may be injected into the tumor area (in the case of solid tumors) and gradually functionalized nanoparticles will be released. Due to its functionalization, they will be attracted mainly to diseased cells, nanoparticles will be biodegraded and drug will be released surrounding the cell. There are several advantages of using combined systems. For example, the highly localized application by avoiding the bloodstream, in the case of solid tumors; an improved release profile with a slow and steady dose that highly favored the drug's effectiveness in eliminating cells. The effectiveness of the functionalization of nanoparticles will be favored by the close application to malignant cells; in general, we will expect to acquire treatments where the typical collateral damage observed in common chemotherapies would be considerably lower as well as the required dose.

#### **6. Acknowledgment**

We want to express our gratitude to Alejandro Martinez and Jorge A. Perez Leon for access to their laboratory and fluorescent microscope supported by CONACYT, Selene Sandoval for sample preparation, Rodolfo Avitia for language assistance and corrections, the University of Arizona and the Autonomous University of Juarez City (UACJ) for the backup and the financial support from PROMEP (Programa para el mejoramiento del profesorado) grant UACJ-EXB-131 and NSF/NIRT grant 0303863.

### **7. References**

applied chemotherapy as well as looking for battling more efficiently and accurately such disease. Although there are a number of published research focused on this issue, the fact is that so far no systems have emerged to prove conclusively its efficacy, although always positive progress has been shown, each proposed system suffers from certain drawbacks that slows down the progress towards real applications. The disadvantages found in the systems of transport and release of anticancer drugs proposed in recent years can be overcome by taking advantage of each method and combining them into drug delivery systems composed with more sophistication and complexity than their predecessors. This idea has already begun to be implemented by some researchers who have integrated drugloaded nanoparticles on thermosensitive hydrogels (Liu et al., 2007), but these authors have not exploited the alternative of functionalization or multi-functionalization of nanoparticles. So, there is still much room for much improvement in these drug delivery systems and it is where researchers could find novel ideas. For example we can think in to make functionalized or multifunctionalized nanoparticles loaded with a anticancer drug, then incorporate them into a thermosensitive hydrogel. With this proposed system, the hydrogel may be injected into the tumor area (in the case of solid tumors) and gradually functionalized nanoparticles will be released. Due to its functionalization, they will be attracted mainly to diseased cells, nanoparticles will be biodegraded and drug will be released surrounding the cell. There are several advantages of using combined systems. For example, the highly localized application by avoiding the bloodstream, in the case of solid tumors; an improved release profile with a slow and steady dose that highly favored the drug's effectiveness in eliminating cells. The effectiveness of the functionalization of nanoparticles will be favored by the close application to malignant cells; in general, we will expect to acquire treatments where the typical collateral damage observed in common

chemotherapies would be considerably lower as well as the required dose.

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**33**

Wilfrido D. Mojica

*United States of America*

**The Evolving Role of Tissue**

*University at Buffalo, State University of New York*

**Biospecimens in the Treatment of Cancer**

The field of surgical pathology is diagnostic in nature and since its inception, has always supported the treatment of cancer. The manner by which tissues are processed may vary between institutions, but the vast majority utilize formaldehyde as the fixative of choice. This fixative allows for excellent architectural tissue preservation thereby enabling optimal microscopic examination, the foundation from which surgical pathology is based upon. From fixed tissue, cancers have been categorized, sub-typed and features enumerated to help elucidate the differences that exist between them. Classification schemes that include grading and staging systems have been incorporated in their examination and, when coupled with the patient's clinical data, have been useful in prognostication and guidance of follow-up treatment. The development of immunohistochemistry has only served to further ingrain the importance of proper tissue fixation with cancer sample preparation. Yet, despite providing the basis for these invaluable contributions, the approach of fixing tissue biospecimens with formaldehyde has, in recent years, undergone criticism as being a suboptimal means of preservation when molecular analysis is desired from these specimens. Added to this are other pressures to develop novel approaches by which tissue biospecimens can be interrogated. Within the field of surgical pathology, it is becoming recognized that various pre and post fixation factors may contribute to negatively impact the overall integrity of the tissue biospecimen. Outside surgical pathology, the continually decreasing size of core biopsy specimens are minimizing the amount of tissue present for architectural evaluation. In addition, newer treatments are continually being sought in order to further personalize cancer treatment based on the type of tumor present in the tissue specimen. These forces are necessitating a re-evaluation of the entire established process of fixation, and whether alternative methods may exist or be developed that may be more amenable for both diagnosis and biospecimen integrity. In this chapter, the central role of tissue biospecimens for the support and guidance of cancer care are discussed, with an examination on how newer technologies and minimally invasive approaches will change the

landscape by which these specimens will be processed.

**2. The history of tissue biospecimens in cancer treatment**

The examination of tissue through the microscope falls within the modern day purview of surgical pathology. Although this examination is now performed by pathologists, the

**1. Introduction**

## **The Evolving Role of Tissue Biospecimens in the Treatment of Cancer**

## Wilfrido D. Mojica

*University at Buffalo, State University of New York United States of America*

### **1. Introduction**

782 Current Cancer Treatment – Novel Beyond Conventional Approaches

Yu, Tang. (2009). Jagdish Singh.Biodegradable and biocompatible thermosensitive polymer

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based injectable implant for controlled release of protein. *International Journal of*

assessment of nanoscale material containing drugs. *Advanced Drug Delivery Reviews,*

The field of surgical pathology is diagnostic in nature and since its inception, has always supported the treatment of cancer. The manner by which tissues are processed may vary between institutions, but the vast majority utilize formaldehyde as the fixative of choice. This fixative allows for excellent architectural tissue preservation thereby enabling optimal microscopic examination, the foundation from which surgical pathology is based upon. From fixed tissue, cancers have been categorized, sub-typed and features enumerated to help elucidate the differences that exist between them. Classification schemes that include grading and staging systems have been incorporated in their examination and, when coupled with the patient's clinical data, have been useful in prognostication and guidance of follow-up treatment. The development of immunohistochemistry has only served to further ingrain the importance of proper tissue fixation with cancer sample preparation. Yet, despite providing the basis for these invaluable contributions, the approach of fixing tissue biospecimens with formaldehyde has, in recent years, undergone criticism as being a suboptimal means of preservation when molecular analysis is desired from these specimens. Added to this are other pressures to develop novel approaches by which tissue biospecimens can be interrogated. Within the field of surgical pathology, it is becoming recognized that various pre and post fixation factors may contribute to negatively impact the overall integrity of the tissue biospecimen. Outside surgical pathology, the continually decreasing size of core biopsy specimens are minimizing the amount of tissue present for architectural evaluation. In addition, newer treatments are continually being sought in order to further personalize cancer treatment based on the type of tumor present in the tissue specimen. These forces are necessitating a re-evaluation of the entire established process of fixation, and whether alternative methods may exist or be developed that may be more amenable for both diagnosis and biospecimen integrity. In this chapter, the central role of tissue biospecimens for the support and guidance of cancer care are discussed, with an examination on how newer technologies and minimally invasive approaches will change the landscape by which these specimens will be processed.

#### **2. The history of tissue biospecimens in cancer treatment**

The examination of tissue through the microscope falls within the modern day purview of surgical pathology. Although this examination is now performed by pathologists, the

The Evolving Role of Tissue Biospecimens in the Treatment of Cancer 785

Along with hematoxylin and eosin, a number of other stains were specifically developed to help identify cell structure and determine cellular function, but in the process aided to further classify tissues and categorize tumors. Their development was spurred by the fact that, despite the enumeration of histologic criteria published and disseminated about specific diseases and tumors, reproducibility among the growing numbers of physicians that practiced pathology remained a problem. In order to engender consensus, a number of special stains were created through experimentation that would eventually serve to better delineate cellular structure, content and properties. One of these stains, the Periodic Acid Schiff stain, allowed for the detection of the presence of carbohydrate macromolecules, a feature that would prove to be a useful ancillary aid to morphology in the differentiation between different types of tumors. For example, the identification of the Periodic Acid Schiff reaction in tumors known as small round blue cells can be helpful in leading to the correct diagnosis. Positive staining material in rhabdomyosarcomas, Ewing's sarcomas, malignant peripheral neuroectodermal tumors and germinomas can help distinguish them from other tumors with similar histologic features such as desmoplastic small round cell tumors, small cell mesothelioma, and a number of other tumors with small round blue cell morphology (Leuschner, 1996). This stain is particularly helpful in identifying Ewing's sarcoma, due to the fact that over 80% of these tumors have been reported to contain glycogen within their cytoplasm. Other special stains, like the Mucicarmine and Alcian blue stains, were originally used to help characterize cells that secreted mucin, aid in the early differential diagnosis of tumors and determine if they were useful in identification of tumor origin for metastatic tumors (Johnson, 1963). These stains have persevered and have modern day utility in defining poorly differentiated tumors as being or not being adenocarcinoma and in distinguishing adenocarcinomas from poorly differentiated squamous cell carcinomas in the evaluation of lung cancers for the former stain, and as an aid in the detection of intestinal metaplasia in the medical condition known as Barrett's esophagus for the latter stain (Wallace, 2009). Yet another special stain, the reticulin stain, showed initial promise for staining specific extracellular matrix constituents, namely the collagen type III fibers that comprise the stromal network in many different organs. It can be particularly prominent in the liver, as these fibers invest the hepatocytes that make up the hepatic plates, with normal hepatic plates being two cell layers thick or less. Reticulin stains help to define hepatic adenomas and hepatocellular carcinomas from normal liver, subtle changes not so obvious when examined by hematoxylin and eosin stains alone. In hepatic adenomas, they show an expansile growth pattern whereas in hepatocellular carcinoma they demonstrate an increase in trabecular thickness. To this day, the reticulin stain continues to be recommended as part

**2.1 Early tools in the assessment of tissue and tumor biospecimens**

of the evaluation of nodules within the liver (Lennerz et, 2009).

Later, the development of the electron microscope, with its ability to probe sub-cellular structure, proved a boon to tissue diagnostics as it related to cancer treatment. Delivery of the correct diagnosis enabled a treating physician the opportunity to give the most clinically relevant care. Electron microscopy allowed for the visualization of subcellular organelles that could otherwise not be discerned using traditional light microscopy. When tumor tissue was examined at the electron microscopy level, the presence or absence of organelles known to be specific to certain cell types enabled the identification of poorly differentiated tumors whose cell of origin could not, at the time, be properly classified at the morphologic level. The electron microscope provided more definitive characterization of tumor subtypes than